A method for obtaining RNA from Hemileia vastatrix appressoria produced in planta, suitable for transcriptomic analyses.

Appressoria are the first infection structures developed by rust fungi and require specific topographic signals from the host for their differentiation. The ease in obtaining appressoria in vitro for these biotrophic fungi led to studies concerning gene expression and gene discovery at appressorial level, avoiding the need to distinguish plant and fungal transcripts. However, in some pathosystems, it was observed that gene expression in appressoria seems to be influenced by host-derived signals, suggesting that transcriptomic analyses performed from in planta differentiated appressoria would be potentially more informative than those from in vitro differentiated appressoria.

Validation of RT-qPCR reference genes for in planta expression studies in Hemileia vastatrix, the causal agent of coffee leaf rust.

Hemileia vastatrix is a biotrophic fungus, causing coffee leaf rust in all coffee growing countries, leading to serious social and economic problems. Gene expression studies may have a key role unravelling the transcriptomics of this pathogen during interaction with the plant host. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the golden standard for gene expression analysis, although an accurate normalisation is essential for adequate conclusions.