Discovery of pathogen-regulated small RNAs in plants.

Department of Plant Pathology, Center for Plant Cell Biology and Institute for Integrative Genome Biology, University of California-Riverside, Riverside, California, USA.

Methods in enzymology 2007

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Small RNAs have emerged as one of the most important regulators for gene expression in eukaryotes. Small RNA-mediated gene silencing has been shown to play an essential role in antiviral defense in both plant and animal systems (Li and Ding, 2005; Voinnet, 2005; Wang et al., 2006). These viral RNA-generated small interfering RNAs (siRNAs) are extragenomic in origin. Studies from our lab and others suggest that host-endogenous small RNAs also play an important role in plant defense in response to other pathogens besides viruses (Katiyar-Agarwal et al., 2006; Navarro et al., 2006). The methods described here provide an opportunity to identify many more novel pathogen-regulated small RNAs in plants, which will help in understanding the regulatory mechanism of plant immunity. Here, we introduce the approaches of powerful high-throughput parallel sequencing and hybridization-based technologies for the discovery and detection of pathogen-regulated small RNAs. We mainly compare and discuss the methods of low-molecular-weight (LMW) RNA extraction from pathogen-infected tissue and strategies for detecting endogenous small RNAs by Northern blot analysis.