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

- Hemileia vastatrix can differentiate appressoria in vitro in oil:collodion membranes.
- In vitro appressoria differentiate at similar rates as in planta appressoria.
- Nail polish replicas can remove intact appressoria from coffee leaf surface.
- Polish-collected appressoria RNA is of good quality and suitable for cDNA synthesis.
- cDNA from polish-collected appressoria can be used 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. Nevertheless analysing appressorial RNA obtained from in planta samples is often hampered by an excessive dilution of fungal RNA within plant RNA, besides uncertainty regarding the fungal or plant origin of RNA from highly conserved genes. To circumvent these difficulties, we have recovered Hemileia vastatrix appressoria from Arabica coffee leaf surface using a film of nitrocellulose dissolved in butyl and ethyl acetates (nail polish), and extracted fungal RNA from the polish peel. RNA thus obtained is of good quality and usable for cDNA synthesis and transcriptomic (quantitative PCR) studies. This method could provide the means to investigate specific host-induced appressoria-related fungal pathogenicity factors.