Small RNA Extraction and Quantification of Isolated Fungal Cells from Plant Tissue by the Sequential Protoplastation

Small RNA (sRNA)-mediated RNA interference (RNAi) is critical for regulating both host immunity and pathogen virulence. Recent studies have revealed that RNA-silencing signals travel between different organisms and trigger gene silencing in trans, termed cross-kingdom RNAi. To investigate cross-kingdom RNAi, it is necessary to purify the fungal cells of interest from infected plants. Here, we present a method for small RNA extraction and quantification of isolated Botrytis cinerea cells from infected Arabidopsis leaves, by utilizing the differences between plant and fungal cell wall components (sequential protoplastation method). The isolated fungal cells are free of contaminants from the host plants, and remain viable, providing high-quality RNA for library construction. This method can be modified to isolate the infection structures of many other plant pathogens from plant tissue.