Project 4: Plant protection on demand

Exudate-driven activation of novel antimicrobials in seed- and root-associated microorganisms





The benefit of plant-actinomycete interactions

Actinomycetes are Gram-positive filamentous bacteria that are abundant in soil and on/in plant roots. They produce a wealth of natural products, including two-third of the known antibiotics (Hopwood, 2007). The in silico mining of Streptomyces genomes suggested that their potential as producers of natural products is much larger than originally appreciated (Challis and Hopwood, 2003). As yet, however, this potential reservoir of active compounds remains untapped because the conditions for their expression remain unknown. The control of so-called cryptic antibiotics is most likely tightly tied to the competitive ecological conditions in which antibiotic production evolved (Cornforth and Foster, 2013). Plants promote symbiosis with actinomycetes and Bacilli to exploit their secondary metabolite repertoire to defend their environment against microbial pathogens (Coombs and Franco, 2003; Ezra et al., 2004; Huang et al., 2011; Lin et al., 2012; Taechowisan et al., 2005). Additionally, endophytic actinobacteria also promote the plant defence systems, allowing plants to respond quickly when challenged (Conn et al., 2008). We recently obtained proof of concept that the plant-defense hormone jasmonate activates antimicrobial production in actinomycetes (Fig. 4), supporting the concept that plant-microbe interactions specifically elicit the production of silent antibiotics to enforce protection at the time of challenge by pathogenic microbes. Here we focus on the interaction between symbiotic actinomycetes and selected Bacillus species and crop plants and in particular the signals (plant exudates, hormones) that elicit production of antimicrobials in spermosphere- and rhizosphere-associated actinomycetes and rhizospheric and endophytic Bacilli, to improve plant defense against biotic stress.

Aims of the project

i) to identify actinomycetes and Bacilli associated with crop seeds and roots that produce antimicrobials specifically in response to plant hormones or exudates and ii) to catalogue the antimicrobials that are produced specifically in response to plant-derived eliciting signals. This will be applied to screen for novel antimicrobials and to obtain a collection of actinomycetes and Bacilli that act as biocontrol agents by producing antimicrobials on demand.

Research Plan

Workpackage 4.1. Plant-secreted compounds that activate antimicrobial production by actinomycetes

Chemical interactions between plants and actinomycetes (and in selected cases isolated Bacillus species) will be analysed as an approach to identify plant-derived elicitors for antibiotic production. An Actinomycete Reference Panel will be used as test system, consisting of 20 well-characterised actinomycetes, namely 18 unpublished isolates from our in-house collection together with Streptomyces coelicolor and S. griseus. Mass spectrometry (MS) and bioactivity profiles for many of the secondary metabolites are known, and their genomes sequenced. Plant hormones (e.g. jasmonate, salicylic acid, ethylene and auxins) and exudates

 (a) Direct metabolite profiling. Hormones and other seed/root exudates (project 1) will be tested for their effects on secondary metabolite and bioactivity profiles of the Reference Panel, both individually and community-wise. Comparison of the MS profiles will give insight into which (classes of) secondary metabolites are elicited by which compounds or extracts and how this correlates to the expression profiles under routine growth conditions. In this way we will catalogue the antimicrobial production response in relation to plant signals.

(b) Biosensors. The genes for green fluorescent protein (eGFP) and the lux operon (encoding luciferase) will be used as reporters and therefore brought under the control of the regulatory regions for known and silent secondary metabolite genes, obtained from the Reference panel. A number of such constructs is already available in our laboratory. Actinomycetes harbouring one of the reporters will either be grown near plant seeds or treated with compounds or extracts, and the responses assessed.

(c) Imaging MS and nanoDESI. Small molecules secreted by actinomycetes during interaction with plant seeds will be visualised using imaging and nanoDESI MS (Watrous et al., 2012), to establish which molecules are received and produced during interaction. IMS will be done in close collaboration with NIOO (project 1 in the program). The data will be compared to those for reporters and MALDI MS profiles, so as to paint a detailed picture of the chemical responses elicited from the interactions. The most promising plant-secreted elicitors obtained via these approaches will then be worked out further, by obtaining related compound families from commercial compound libraries. Where required, compounds can be further derivatized using bioorganic chemistry in the group of our collaborator Prof. Hermen Overkleeft in Leiden. The optimized elicitors are ideal new means to screen other actinomycetes isolated from plant seeds and roots (project 1) for novel antimicrobials.

Workpackage 4.2. Competition-mediated activation of antimicrobials

Signals transmitted by specific (competing) bacteria have a major impact on natural product formation by actinomycetes (Garbeva et al. 2011; Watrous et al., 2012). Interaction of actinomycetes with Bacillus and Pseudomonas species will be a focal point. Based on the metagenomic/transcriptomic analyses of the microbiomes of seeds and roots of crops (see projects 1-3), actinomycetes and Bacilli will be isolated. The communities will be classified using a Bruker Biotyper and 16S rRNA sequencing. The composition of a control treatment with only media will be compared to that of different treatments to which various plant hormones and derivatives are added, at different concentrations. Species that are increased in density by plant-derived compounds are candidate antibiotic producers and will be tested further in situ. Consortia of different seed and root isolates will then be grown on agar plates and the effects of plant hormones on the composition of the community studied. The actinomycetes that stand out from the competition experiments will be studied as described above in project 4.1. Spermosphere- or rhizosphere-derived actinomycetes that produce antimicrobials on demand by plants will be identified and application in root colonization and bioprotection of the different crop species against biotic and abiotic stress analyzed in close collaboration with partners in projects 2 and 3. The genome mining tool Bagel3 (van Heel et al., 2013) will be employed by the Kuipers group (RUG) to identify novel antimicrobial gene clusters in the isolated microbiota.

Workpackage 4.3. Compound Identification.

Antimicrobial activities will be assessed against a routine set of bacterial and fungal indicator strains (Bacillus, E. coli for antibacterials, Fusarium and Aspergillus for antifungals). We routinely screen compounds against a range of multi-drug-resistant (MDR) pathogens, which can be done in the medical centres of Leiden University and Erasmus University (Rotterdam). The latter involves an intensive collaboration with the clinical microbiology laboratory led by Dr. van Wamel. Candidate compounds will be dereplicated by LC/MS/MS against databases of microbial natural products (ChemSpider, Chabman Hall Dictionary of Natural Products and AntiBase) and to obtain the molecular formula. 1D and 2D NMR (COSY, ROESY, TOCSY, NOESY, HMBC, HSQC) will provide the structural information. When needed, structural information will be obtained by X-Ray crystallography. To rapidly identify gene clusters specifying a natural product of interest, we  developed a quantitative proteomics-based method, by correlating the level of bioactivity under a number of different growth conditions to the global proteome expression profiles (Gubbens et al., 2014). This proteomining approach will be applied to identify the gene clusters for promising compounds, which is an important step towards further optimization of expression and exploitation. Lead compounds will be commercialized in conjunction with the users.




Problems and economic value

As a consequence of the overuse of antibiotics in the 20th century, many multiple drug resistant bacteria have emerged in all parts of the world. These include MDR- and XDR-TB (multiple and extremely drug resistant tuberculosis, respectively), MRSA (methicillin-resistant S. aureus), PRSP (penicillin-resistant Streptococcus pneumoniae), and VRE (vancomycin-resistant Enterococcus). Perhaps the biggest threat to human health is MDR-TB, and the last report of the WHO (26th of Feb 2008) sends another warning, estimating that of all TB cases now around 10-15% is a multidrug resistant variant (around 5% of new cases and around 30% of the previously treated cases). Clearly, there is a strongly increasing demand for the discovery and production of novel anti-infectives. Considering this enormous threat to human health, new research lines focused on generating novel antibiotics are required. In recent years, much attention has been directed towards silent antibiotics, i.e. those that are poorly expressed under routine growth conditions. Exploiting this treasure trove is the major challenge in current antibiotic research. The total market for clinical antibiotics is around 40 billion dollars annually. Similarly in agriculture and horticulture there is a strong need for new antimicrobials, due the emergence of fungicide resistance in pathogen populations and concomitant changes in regulation of pesticide applications. These problems necessitate the need for more sustainable and environment-friendly solutions.


Through this proposal we will address the requirement for antimicrobials for crop protection, and at the same time exploit the power of plant-microbe interactions as a natural elicitor of silent and poorly expressed antibiotics. By identifying Actinomycetes and Bacilli in the spermosphere and rhizosphere of plants, and selecting those that specifically produce antibacterials and antifungals on demand by plants (i.e. crops), we will generate novel biocontrol agents. Furthermore, the insights obtained in terms of plant-derived signals that activate antibiotics and antifungals will be applied to screen directly for antimicrobials in culture collections uniquely available to the consortium, with approaches as recently described (Zhu et al., 2014). Together, this will generate two different types of products, namely antimicrobial-producing actinomycetes and Bacilli for use in protective soils, and antimicrobial compounds for use in the clinic or in food preservation.

Translation and implementation

As soon as promising antimicrobials (hits) have been identified, they will be tested for application as antibiotics (cytotoxicity, stability, target host range etc). BDS/MLS has the tools and infrastructure for tox screening. The groups of van Wezel and Kuipers have ample experience with identifying and developing antimicrobials, among others within the GenBiotics program, a former and very successful Perspectief Program involving a large network of academic institutions and biotech companies. Our industrial partners DSM and Corbion are major players in the pharmaceutical and/or food industries, and this is the most obvious and direct route to market. For application of the actinomycetes identified as potential biocontrol agents (as producers of antimicrobials in the rhizo- and spermosphere) we have leading Dutch plant breeding industry on board, and further marketing will be discussed with these end users.


TLC biogram (left) and proton NMR profiles of secondary metabolites produced by Streptomyces sp. MBT3 grown under different conditions.
Lanes: 1, without plant exudates; 2, with soil extract; 3, challenged with jasmonate (JA); 4, challenged with salicylic acid.
Note the strong induction of antimicrobial activity (visible as a zone of clearance on the TLC biogram and peaks indicated by red arrows in the NMR spectrum).
The TLC biogram was obtained by separating extracts using TLC followed by overlay of the plate with topagar containing Bacillus subtilis.