Dissecting the Subnuclear Localization Patterns of Argonaute Proteins and Other Components of the RNA-Directed DNA Methylation Pathway in Plants

RNA-directed DNA methylation (RdDM) is a nuclear pathway which is comprised of multiple main and accessory protein components, including two plant-specific DNA-dependent RNA polymerases, Pol IV and Pol V, and argonaute (AGO) proteins. Regulation in the RdDM pathway can be achieved via multiple mechanisms, including the spatial distribution of different RdDM components. Here we describe a protocol for dissecting the subnuclear localization of AGO proteins and other RdDM components, including nuclei extraction from seedlings, slide preparation, and subsequent immunostaining.