In this work package, we will focus on the interaction between CLPs and plant roots. This interaction will be studied at three different levels, namely the phenotypical, molecular and mechanistic level referring to the ability of CLPs to induce systemic resistance, to trigger transcriptomic and metabolic changes in roots and to interact specifically with membranes, respectively.
Although some CLPs have potential to trigger induced systemic resistance, no extensive studies on CLP-ISR have been performed. Here we will study if the purified/synthetic CLPs, selected based on the outcome of WP1 and including different CLP families and variants, are able to induce a systemic response and to protect plants against subsequent fungal attack. Plant roots treated with CLPs will be assessed for their susceptibility against these leaf pathogens. If CLPs are able to trigger ISR via their interaction with roots, one can expect significant alterations in root gene expression and metabolites. Conversely, some CLPs may not play any role in ISR-mediated signalling but may rather be involved in host suppression for efficient bacterial colonization.
Analysing the expression levels of several marker genes and/or ROS production over a period of time will give an indication at which time points the plant’s physiological and metabolic processes are modulated and/or when immune responses are activated. Through correlation analyses, changes in the root transcriptome will be coupled to changes in root metabolome. This will further assist in linking metabolites to genes, and to reconstitute pathways that play an important role upon CLP treatment.
On another hand, we have accumulated strong evidence showing that CLPs preferably interact with the lipid phase of the plasma membrane rather than via perception by specific PRR as usually observed for most MAMPs. Zeta potential measurements, ITC, and time-resolved fluorescence of calcium sensitive dyes will be used to elucidate this behaviour in detail. Finally, the correlations will be assessed between calcium effects in model systems and in vivo, particularly the Ca2+ spike in CLP-treated cells. Further insights into the mechanistics of CLP – plasma membrane lipid interactions will be obtained by exploiting various biophysical assays such as vesicle leakage experiments (fluorescence lifetime) to appreciate the effect of non-lethal challenges on plant membrane integrity and zeta potential measurements that detect the translocation of the CLP itself. In addition, direct measures of domain formation will be pursued including the detection of phase separation in suitable model membranes by differential scanning and pressure perturbation calorimetry (DSC and PPC). Furthermore, formation and alteration of nano-sized membrane domains will be detected by time-resolved Förster resonance energy transfer (FRET) between membrane-bound fluorophores (66). NMR investigations in CLP model membrane