Maintenance and Quantitative Phenotyping of the Oomycete-plant Model Pathosystem .

The interaction between the host plant and the oomycete is an established model system for the study of an obligate biotrophic downy mildew interaction. The evaluation of the developmental success of 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 bioassay to provide a practical guide for the initial setup of this system in the laboratory. This pipeline includes the following steps: A) growing , B) propagation and strain maintainance C) 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 and , the protocol steps B-E should be easily adjustable for the study of other plant-oomycete pathosystems.