Background

Powdery mildew of cereals, caused by Blumeria graminis, leads to significant yield loss (Spanu et al., 2010). The barley powdery mildew fungus B. graminis f. sp. hordei, is economically important, and one of the best studied powdery mildews (Both et al., 2005a; Bindschedler et al., 2009). This fungus is an obligate biotroph, which means it can only infect and propagate on living barley plants (Pedersen et al., 2012). Its asexual life cycle involves an intimate relationship with the host plant. Airborne conidia germinate on the host producing first a primary and then a secondary germ tube. The secondary germ tube develops an appressorium, from which a hyphal peg penetrates into an epidermal cell. Inside the cell, a penetration peg enlarges and forms a multidigitate feeding structure: the haustorium. This is surrounded by a plant-derived extrahaustorial membrane (Both et al., 2005b; Bindschedler et al., 2016).

Several studies of B. graminis f. sp. hordei have characterized gene expression profiles and proteomics of this fungus (Bindschedler et al., 2011; Pennington et al., 2016). However, the obligate biotrophic nature of B. graminis poses exceptional challenges to the investigations of this fungus, especially the studies of haustoria. The intracellular haustorium and the extrahaustorial membrane constitute a special compartment, which is functionally essential for the interaction with the host (Wang et al., 2009; Pliego et al., 2013). Investigating haustoria is critical for further understanding the mechanism of nutrient uptake into the pathogen as well as plant-pathogen recognition. Therefore, obtaining purified haustoria from infected plants has the potential of contributing significantly to these studies.

Previously, the procedure for isolating haustoria from obligate biotrophs employed affinity chromatography on rust fungi (Catanzariti et al., 2006; Garnica et al., 2014). During the 1990s, several centrifugation-based methods were introduced to isolate powdery mildew haustoria from pea plants (Mackie et al., 1991; Testut et al., 1999). This method was then applied to powdery mildews of other plants, including Arabidopsis (Wang et al., 2009; Micali et al., 2011). More recently, a filtration and gradient centrifugation-based method was described to isolate powdery mildew haustoria from barley (Godfrey et al., 2009). The hallmark of these published methods is that they are relatively laborious and require several different kinds of isolation buffers, different sizes of steel meshes and multiple centrifugation steps. Besides, the isolation procedure was required to be performed on ice. In this protocol, we developed a simpler way to isolate B. graminis haustoria from infected barley leaves. Releasing of haustoria from plant cells is achieved by enzymatically degrading the epidermal cell walls, and the purification of haustoria by filtration through a nylon mesh. This method requires digestion of the host plant cell walls, a single filtration and one centrifugation step.