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Cuscuta spp. are widespread obligate holoparasitic plants with a broad host spectrum. Rootless Cuscuta penetrates host stems with so called haustoria to form a direct connection to the host vascular tissue (Dawson et al., 1994; Lanini and Kogan, 2005; Kaiser et al., 2015). This connection allows a steady uptake of water, assimilates and essential nutrients from the host plant and therefore enables Cuscuta growth and proliferation. To quantify the parasites’ ability to grow on potential host plants one can use the quantitative growth assay (Hegenauer et al., 2016) described herein, which exclusively utilizes fresh weight measurement as readout.

The production of 'reactive oxygen species' (ROS), also termed oxidative burst, is a typical cellular response of animals and plants to diverse biotic and abiotic stresses. Here, we outline the detection of the ROS-burst in plant leaf pieces using a luminol-based bioassay which allows for the detection of chemiluminescence. The assay was originally described by Keppler et al. (1989) and subsequently adapted for other plant cells and tissues (Felix et al., 1999) and also used in recent publications (Albert et al., 2013; Albert et al., 2010; Butenko et al., 2014; Halter et al., 2014). In this protocol we outline a standardized version of this assay including remarks and recommendations for data evaluation and interpretation of results.

Studying the biochemical interaction of ligands with their corresponding receptors requires highly sensitive detection and monitoring of the bound ligand. Classically, radioactively labelled ligands have been widely used as highly sensitive tools for such binding measurements. Disadvantages of radiolabelling include instability of products, high costs and risks of working with radioactivity. Thus, assays using chemiluminescent probes offer convenient, highly sensitive alternatives. Here we suggest acridinium esters as suitable conjugates to label ligands of interest. Chemical oxidation of acridinium esters triggers chemiluminescence, allowing quantitation of this compound down to amol concentrations in standard luminometers. The first report about acridinium esters in immunoassays date back to 1983 (Weeks et al., 1983) and demonstrated the ability to conjugate acridinium to peptides, followed by using such peptides to measure receptor – peptide ligand interactions (Joss and Towbin, 1994). Recently, this binding assay was adapted for studying derivatives of the plant peptide IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) and their interaction with the corresponding receptor HSL2 (HAESA-LIKE 2) was reported (Butenko et al., 2014). Here we describe how this sensitive, nonradioactive binding approach can be used to reveal receptor-ligand binding in plant material.