Abstract
The interaction between the host plant Arabidopsis thaliana (Arabidopsis) and the oomycete Hyaloperonospora arabidopsidis (Hpa) is an established model system for the study of an obligate biotrophic downy mildew interaction. The evaluation of the developmental success of Hpa is often based on the quantification of reproductive structures that are formed on the surface of leaves, such as the sporangiophores or the conidiospores they carry. However, the structural basis of this interaction lies within the plant tissue and, in particular, the haustoria that form inside plant cells. Therefore, valuable additional information about the performance and compatibility of the downy mildew interaction can be gained by light microscopical inspection of the hyphal and haustorial shape inside the plant tissue and within plant cells respectively. Here we describe a protocol for the visualization and quantification of morphological phenotypes inside the plant. While we focus specifically on the quantification of haustorial shape variants, the protocol can easily be adapted for the quantification of other morphological features such as hyphal deformations, or oogonia frequency. By including and refining already existing protocols from a variety of sources, we assembled the entire experimental pipeline for the Arabidopsis Hpa bioassay to provide a practical guide for the initial setup of this system in the laboratory. This pipeline includes the following steps: A) growing Arabidopsis, B) Hpa propagation and strain maintainance C) Hpa inoculation and incubation D) staining of plant tissues for visualization of the pathogen and E) an introduction of the Keyence VHX microscope and Fiji plugin of ImageJ for the quantification of structures of interest. While described here for Arabidopsis and Hpa, the protocol steps B-E should be easily adjustable for the study of other plant-oomycete pathosystems.
Keywords: Arabidopsis, Hyaloperonospora arabidopsidis, Trypan blue staining, Haustoria quantification, Multilobed haustoria, Plant-microbe interaction
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.
Materials and Reagents
Equipment
Software
Procedure
Recipes
Acknowledgments
The initial establishment of the Hpa x Arabidopsis pathosystem in the author’s laboratory was largely based on Hpa genotypes and advise given by the team of Jane Parker, while still working at The Sainsbury Laboratory in Norwich. The Steps B1 and D described in this protocol are similar to the one described by Asai et al. (2015). Procedures A-D were used as described in Ried et al. (2019). MP acknowledges funding by the Deutsche Forschungsgemeinschaft (DFG) and the European Research Council (ERC).
Competing interests
The authors have no competing interests to declare.
References
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