Objectives

CLP selection based on interactions of the producing strains with host plant and fungal pathogens

Selecting CLPs based on their efficient production under in vitro conditions in rich artificial medium is by far much less relevant than considering CLPs that are actually formed by cells growing on roots and/or feeding on exudates. Therefore, the objective of this first WP is to develop new approaches and to exploit new analytical technologies and cultivation methods to setup relevant ex-vivo systems for the study of CLP production by bacterial cells interacting with their plant host and other microorganisms (fungi and other CLP producers).

This can be achieved by identifying in situ produced CLP and by resolving the dynamics of production of the various CLPs released by each strain colonizing roots. Generating root exudates from different plant species with the aim to correlate CLP production with the one observed in planta will allow to create an Exudate Mimicking Medium (EMM) that will be used throughout the project. The rationale in using EMM for these interactions is also to keep with a “plant nutritional context” that may be of crucial influence regarding growth and physiology of microbial partners.

Role of CLPs in colonization and biofilm formation

Efficient root colonization means successful establishment in the ecological niche, which is also a prerequisite for biocontrol of phytopathogens. This process is considered to rely partly on the ability to move on the root surface and to form consistent biofilms. The objective of this WP is therefore to demonstrate that the potential to produce specific CLPs favours motility, biofilm and hence early colonization of plant roots and to determine which CLP families/variants identified in WP1 as being produced on roots are necessary for those processes.

CLP-dependent motility in wild-type co-producers will be assessed using typical swarming assays on soft agar EMM combined with IMS in order to correlate dynamics of the swarming front cells with the accumulation of a particular CLP in the corresponding area. Afterwards, the role of specific CLPs in biofilm formation will be established in more detail by combining various chemical and biological approaches.

CLP-mediated interactions between micro-organisms

The CLPs produced in situ in the presence of pathogenic fungi (WP1) will be selected to determine their individual and synergistic contribution to fungal antagonism. In addition, the potential physical interaction between in planta produced CLPs will be studied.

The observation that physical interactions between CLPs can occur, as exemplified by the so-called “white line reaction” between CLP producers and between CLP producers and fungi in agar media, raises the question whether such interactions may also play a role during colonization of the rhizosphere. In WP1, mass spectrometry imaging analysis of roots co-inoculated with WLR-positive partners (and defined mutants) can show whether such combination has any effect on root-associated CLP production. For bacterial combinations showing competitor-modulated CLP production in WP1, mass spectrometry imaging will be used to monitor the dynamics of CLP production when bacteria are confronted on EMM agar medium. For such experiments, study of dynamics of the WLR irrespective of its biological significance, is a suitable model system (with a visually observable phenotype). Control experiments will involve mutants that are affected in the individual CLPs. At present, it is not known whether additional factors may be required in the apparent coprecipitation of CLPs but mass spectrometry imaging analysis has the capacity to reveal such additional interaction partners.

CLP-mediated interactions with plants

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

CLP origin and diversity

Here, we will integrate the knowledge generated by chemical characterization of CLPs with genomic analysis of the CLP biosynthetic systems assigned to taxonomic phyla. In addition to expanding CLP diversity and providing insight on how this diversity evolved, this information will serve to propose an optimized classification for bacterial CLPs produced by Pseudomonas and Bacillus. For those CLPs studied in more detail in the previous WPs, we will also link structure with function. Progress made in the genetic and chemical (structural) characterization of CLPs enables to take a bird’s eye view on the huge diversity of the bacterial metabolites and may be used to design a hierarchical classification system, which may subsequently be annotated with functional information.

Insight in how the huge CLP chemical diversity has evolved can be generated from systematic phylogeny-based comparison based on the sequence information contained in NRPS-encoding genes, in particular the substrate-recruiting A-domains. Genomic sequence information of bacteria (including large numbers of bacilli and pseudomonads) represents a vast amount of information on the CLP-assembling NRPS systems that is largely unexplored. By genome mining, this raw information can be retrieved, analysed and used to detect evolutionary relationships between NRPS systems that are not readily apparent by comparison of chemical structures of the corresponding CLPs.