Specific Isolation of RNA from the Grape Powdery Mildew Pathogen Erysiphe necator, an Epiphytic, Obligate Parasite

RNA expression profiling of obligately parasitic plant microbes is hampered by the requisite interaction of host and parasite. This can be especially problematic in the case of powdery mildews, such as Erysiphe necator (syn. Uncinula necator), which grow superficially but tightly adhere to the plant epidermis. We developed and refined a simple and efficient technique in which nail polish was used to remove conidia, appressoria, hyphae, conidiophores, and developing ascocarps of E. necator from grapevine (Vitis vinifera) leaves and showed that RNA isolated after removal was not contaminated with V. vinifera RNA. This approach can be applied to expression analyses throughout fungal development and could be extended to other epiphytic pathogens and saprophytes. Introduction Powdery mildews are caused by obligately parasitic fungi in the Erysiphales that superficially colonize plant hosts and are among the most economically important and widespread plant pathogens (Bélanger et al., 2002). In spite of their importance, molecular characterization of powdery mildews is poor, with the notable exception of Blumeria graminis, the sole species of a phylogenetically distinct tribe of powdery mildews whose members infect grasses (Glawe, 2008). Aside from B. graminis, only 23 expressed sequence tags (ESTs) have been deposited at GenBank for the Erysiphales (National Center for Biotechnology Information, 2009). A major contributing factor to this paucity of expression data for powdery mildews is the inability to isolate and maintain the fungus in axenic culture. By contrast, for another economically important ascomycete, Botryotinia fuckeliana, which can be cultured axenically, 10982 ESTs have been deposited at GenBank (National Center for Biotechnology Information, 2009). In general, powdery mildew pathogenesis begins by deposition of ascospores or conidia onto susceptible host tissue, initiating an infection process (Pearson and Goheen, 1988). In Fig. 1a, this is depicted by fluorescence microscopy of Erysiphe necator (syn. Uncinula necator), causal agent of grape powdery mildew. Germination and appressorium formation are hostindependent processes that can occur on inert surfaces (Ficke et al., 2003). However, infection requires successful penetration of the host cuticle and cell wall under the appressorium, and establishment of a haustorium (the feeding structure) in a host epidermal cell (Pearson and Goheen, 1988). Following formation of the haustorium, hyphae will elongate, branch, and form additional appressoria, each potentially giving rise to haustoria (Fig. 1a). Thus, with the exception of the haustoria, the pathogen is wholly external to the host. Several days after infection, conidiophores and conidia are produced (Fig. 1b). When hyphae of opposite mating types adjoin, sexual reproduction occurs and chasmothecia (syn. cleistothecia), the overwintering structures that contain ascospores, are initiated (Fig. 1c). Sexual reproduction of E. necator typically occurs late in the growing season (Gadoury and Pearson, 1988). To obtain powdery mildew DNA free of contaminating plant DNA, fungal tissue can be harvested from infected plant tissue by vacuum (conidia) or by scraping (conidia and some mycelia) prior to DNA isolation (McDermott et al., 1994; Jankovics et al., 2008). Such approaches have been used to develop rando, amplified polymorphic DNA and amplified fragment length *Equal first authors. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. J Phytopathol 158:69–71 (2010) doi: 10.1111/j.1439-0434.2009.01578.x Published 2009. This article is a US Government work and is in the public domain in the USA.