Background

Downy mildews are a group of phytopathogenic oomycetes characterized by branched sporangiophores that protrude through leaf stomata, giving the appearance of a white to grey “down” on the affected tissues (Agrios, 2005). Hundreds of Downy mildew species have been described on a wide variety of host plants including monocotyledon and dicotyledon crops (Callan and Carris, 2004). They are typically obligate biotrophs, meaning that they have not yet been successfully cultured outside the plant host with completion of their life cycle. A model system to study the molecular and genetic determinants underlying the downy mildew disease is the interaction between the host plant Arabidopsis thaliana and its oomycetal pathogen Hyaloperonospora arabidopsidis (Hpa). Downy mildew oomycetes typically form a structure called haustorium inside the plant host cell. The haustorium is surrounded by a plant cell membrane called extrahaustorial membrane (EHM) that prevents direct contact of the oomycete with the plant cytoplasm. While the hypothetical role of the haustorium in nutrient uptake from the plant host remains to be confirmed, it probably represents a platform for delivering oomycetal effectors to plants (Judelson and Ah-Fong, 2019) which enable the establishment and maintenance of a biotrophic interaction with the host. It is therefore not surprising that several proteins required for the resistance against Hpa are localized to the EHM (Wang et al., 2009; Caillaud et al., 2014). Despite the obvious relevance of the haustorium for the downy mildew interaction, surprisingly few studies evaluated the interaction by phenotyping the haustoria. A method for the inoculation of Arabidopsis by Hpa was described by Asai et al. (2015). Here we expanded this protocol resulting in a step-by-step guide for the study of this pathosystem in the lab. This includes details on how to sow and grow Arabidopsis, how to propagate Hpa, and specific details on how to systematically record structural features of this interaction with the Keyence VHX digital microscope, and quantify them with the image analysis software Fiji, here exemplified by the quantification of haustoria-shape variation. Our step-by-step guide for the microscopic analysis of the Hpa-Arabidopsis interaction should be easily adaptable for the use not only in other downy mildew interactions in host species other than Arabidopsis but also other hyphal pathogens other than oomycetes. The steps that most likely need attention when moving to other species combinations are highlighted in the protocol below.