This thesis aims to investigate the effect of tripartite interaction between microbial inoculants, the plant, and herbivore insects on the rhizosphere microbiome and volatilome. We investigated the... Show moreThis thesis aims to investigate the effect of tripartite interaction between microbial inoculants, the plant, and herbivore insects on the rhizosphere microbiome and volatilome. We investigated the rhizosphere microbiome and volatilome of tomato plants exposed to insect herbivory and/or inoculated with beneficial microbes known to trigger ISR. First, we reviewed the abiotic and biotic factors that impact the success of ISR microbial inoculants (Chapter 2). Then, we tested microbial inoculants against different stresses and experimental conditions to compare interactions in variable contexts (Chapter 3). Next, we explored the impact of insect herbivory aboveground, on the volatile and microbial belowground compartment. In Chapter 4 we explored root volatiles under stress in two tomato species to evaluate the genotype impact on the stress-induced root volatilome. In Chapter 5, we studied the impact of endosymbiotic fungi arbuscular mycorrhizal fungi (AMF) on root volatiles in an in vitro bioassay and in a greenhouse setup with herbivory-stressed plants. In Chapter 6, we compared the effect of four phylogenetically diverse bacteria and fungi, inoculated as single-species and as a synthetic community, on the rhizosphere microbiome assembly and volatilome in herbivory-stressed plants. Overall, this thesis delves into overlooked interactions providing novel data on belowground plant-microbe interactions. Show less
Polymyxins are clinically used antibiotics, discovered in mid-20th century. Once abandoned due to excessive nephrotoxicity, they are now used increasingly to address infections caused by multi-drug... Show morePolymyxins are clinically used antibiotics, discovered in mid-20th century. Once abandoned due to excessive nephrotoxicity, they are now used increasingly to address infections caused by multi-drug resistant Gram-negative bacteria.In this thesis, we describe the development and synthesis of analogues of polymyxin, aimed at reducing its associated nephrotoxicity. Analogues were made by semisynthesis, with modifications introduced mostly in the exocyclic portion of the molecule. Especially the introduction of a disulfide bond within the linked lipid helped in reducing the toxicity of the molecules, as evidenced by testing on proximal tubule epithelial cells. For most potent analogues, the antimicrobial activity was completely retained.In addition, this thesis describes studies on the mechanism of action of polymyxin, mostly based on the full stereoisomer of polymyxin B4. This analogue lacks antimicrobial activity, indicating its original stereochemistry to be of utmost importance for its use as an antibiotic.Hybrids based on polymyxin B derivatives are described, addressing non-conventional targets. A hybrid with vancomycin (typically active on Gram-positive bacteria only) shows activity on Gram-negative bacteria. A polymyxin-based hybrid coupled to a peptide with a beta-hairpin motif addresses Gram-negative bacteria, presumably by binding to outer membrane protein BamA. Show less
The haplolepideous mosses (Dicranidae) comprise about 4000 species distributed over a wide range of habitats, with great gametophytic and sporophytic morphological variation. Their monophyly is... Show moreThe haplolepideous mosses (Dicranidae) comprise about 4000 species distributed over a wide range of habitats, with great gametophytic and sporophytic morphological variation. Their monophyly is well supported by the results of several molecular phylogenetic studies, which shed light on their relationships and circumscriptions, and thus also contributed to identify many remaining problems. Dicranidae ordinal classification is not congruent with current relationship hypotheses and is not supported by morphology. Morphological circumscriptions of some families do not correspond to monophyletic groups. Furthermore, the monophyly of many families and genera with weak morphological circumscriptions remains to be tested. In this thesis, systematics and relationships of the leucobryoid mosses and some families and genera segregated from the former Dicranaceae s.l. were studied using molecular phylogenetic methods. 37 out of the 38 haplolepideous moss families were represented by markers from the three genomes (nrITS, nad5, trnS-trnF, atpB-rbcL). Phylogenetic reconstructions were based on maximum parsimony, maximum likelihood, and Bayesian inference. Ancestral state reconstructions, phylogenetic network analysis (NeighborNet), and relationship hypothesis testing (Shimodaira-Hasegawa test) were performed to contribute to the interpretation of the results of the phylogenetic reconstructions. Morphological circumscriptions were evaluated and improved whenever possible, in line with the results of all analyses performed. Show less
The plant hormone auxin regulates plant growth and development through polar cell-to-cell transport-generated maxima and minima. PIN FORMED (PIN) auxin efflux carriers determine the direction of... Show moreThe plant hormone auxin regulates plant growth and development through polar cell-to-cell transport-generated maxima and minima. PIN FORMED (PIN) auxin efflux carriers determine the direction of this auxin flow through their asymmetric placement on the plasma membrane (PM). In Arabidopsis thaliana (Arabidopsis), the PM-associated protein kinase PINOID (PID) regulates PIN localisation and thereby auxin transport polarity by phosphorylating these carriers in their central cytoplasmic loop. PID in turn interacts with the calmodulin-like protein TOUCH3 (TCH3) in response to elevated cytosolic calcium, which dissociates PID from the PM and inhibits its kinase activity. In this thesis, we show that PID also interacts with 10 other CALMODULIN/CALMODULIN-LIKE proteins, which together with TCH3 form a confined clade in the CaM/CML family. The CaM/CML binding domain in PID was found to map to an amphipathic alpha-helix inserted in the catalytic kinase core. Disruption of this alpha-helix did not affect PID kinase activity, but impaired both its PM association and the interaction with the CaM/CMLs, making the kinase “untouchable”. Expression of “untouchable” PID versions in the pid mutant background revealed that proper calcium-CaM/CML-PID signalling is essential to maintain the robust spiral phyllotaxis that is typical for the Arabidopsis inflorescence. Show less
Given the accelerating appearance of antimicrobial resistance, there is an urgent need for more fundamental research into novel antibiotic strategies. The work in this thesis helps to address this... Show moreGiven the accelerating appearance of antimicrobial resistance, there is an urgent need for more fundamental research into novel antibiotic strategies. The work in this thesis helps to address this global problem by developing new antibiotic compounds, inspired by the antibacterial mechanisms of the natural antibiotic bacitracin. By unravelling the unique mechanism of action that bacitracin employs, we discovered that the inclusion of a small hydrophobic group in key locations of the molecule results in a dramatic enhancement of antibacterial activity, in some cases more than 100 times more potent than bacitracin. Crucially we found that the most potent analogues are particularly active against antibiotic-resistant bacteria including those bearing clinically challenging resistance genes. In doing so we have developed potent next-generation variants of this classic antibiotic and have taken important steps in the fight against antimicrobial resistance. Show less
In recent decades, climate change has led to more frequent and severe drought events, causing serious consequences such as increased forest mortality and significant crop yield losses.... Show moreIn recent decades, climate change has led to more frequent and severe drought events, causing serious consequences such as increased forest mortality and significant crop yield losses. Understanding how drought affects plants, especially economically important herbaceous species, is crucial for predicting and developing drought-resistant crops. To address this issue, this study analyzed a comprehensive dataset of anatomical and hydraulic traits in different genotypes of Arabidopsis thaliana and tomato, including both wild-type and transgenic mutants. The study also investigated the expression of four well-known drought marker genes associated with ABA-dependent and ABA-independent pathways and the impact of overexpressing the JUNGBRUNNEN1 (JUB1) gene on drought response. The findings revealed that each genotype had a unique set of traits to cope with drought, which could be categorized into two response strategies. One group enhanced their drought resistance through traits like a more negative stem P50, thicker intervessel pit membranes, a more lignified inflorescence stem, and a gradual reduction of the low initial stomatal conductance during drought. This strategy enabled them to maintain a relatively high and stable leaf water potential (Ψl). The second group, represented by JUB1 overexpression genotypes, relied primarily on maintaining a high Ψl which is possibly due to osmoprotectant accumulation in leaves, while the other traits have not been recorded. Overall, this research demonstrated the adaptive capabilities of herbaceous plants to drought conditions, highlighting the intraspecific variation in drought responses that underscores the need for a detailed assessment of drought-responsive traits to improve crop yield in a warming world. Show less
The mycorrhizal symbiosis is among the most widespread species interactions on Earth. This thesis focuses on orchid mycorrhiza, a unique mycorrhizal type that has caught scientists’ attention for... Show moreThe mycorrhizal symbiosis is among the most widespread species interactions on Earth. This thesis focuses on orchid mycorrhiza, a unique mycorrhizal type that has caught scientists’ attention for centuries. By merging the concepts and approaches of molecular phylogenetics, microbial community ecology, and plant ecophysiology, this thesis provides an overview of the evolution and ecology of orchid mycorrhizal interactions from both the fungal and plant perspective, and from the global to the local scale. In this thesis, the variation in fungal associations in space and in time is a central topic, which was inferred by analyses on phylogenetic signals and variation in fungal community structure and also captured by empirical experiments using metabarcoding approaches and quantitative ddPCR. Based on the insights gained in this thesis and that of previous studies, this thesis proposes several directions for future studies to deepen our understanding of the evolution, ecophysiology, and molecular biology of mycorrhizal symbiosis, and thus to benefit orchid conservation and ecosystem restoration. Show less
Songbirds as vocal learners learn their songs and song preference from social tutors. Tutor choice for both song and preference learning are important to characterize for understanding individual... Show moreSongbirds as vocal learners learn their songs and song preference from social tutors. Tutor choice for both song and preference learning are important to characterize for understanding individual learning performance and cultural transmission of song. It is unclear whether early learned song preferences are generalised to songs similar to the tutor song and how simple versus complex early social environment might affect tutor choice and learning contents. This thesis addresses these questions in the zebra finch, the primary avian model for vocal learning studies, by reviewing the existing literature on song and preference learning and conducting three experiments. In these experiments, family raised female zebra finches were tested with a stimulus set of four songs along a gradient of decreasing similarity with the song they learned early in life. A breeding and song learning experiment comparing zebra finches raised with a single versus multiple tutors was conducted. The results suggest that female zebra finches generalise their early learned preferences for a specific song to other unfamiliar songs along a gradient of similarity. Social environment and individual differences can affect tutor choice for both song and preference learning in this colonial species. Show less
This thesis describes how different colours of light affect various aspects of the growth and development of Arabidopsis and tomato plants. In the first chapter, we review the current knowledge of... Show moreThis thesis describes how different colours of light affect various aspects of the growth and development of Arabidopsis and tomato plants. In the first chapter, we review the current knowledge of light receptors and different light-regulated processes. In the second chapter we investigated the effect of direct illumination of roots. In the third chapter we presented an overview of the phenotypes that arise when plants are grown in white, red or blue light. In the fourth and fifth chapter we further investigate the stem and flowering phenotypes that were observed in the different light conditions. Altogether we show that red and blue light often act antagonistically, and that some light-regulated processes may be species- or age-specific. Show less
Striga hermonthica, commonly known as witchweed, infests major cereal crops in Sub-Saharan Africa causing severe yield losses and threatening the livelihood of millions of resource poor farmers.... Show moreStriga hermonthica, commonly known as witchweed, infests major cereal crops in Sub-Saharan Africa causing severe yield losses and threatening the livelihood of millions of resource poor farmers. Despite the use of herbicides, Striga-resistant crop varieties and agronomic practices to mitigate the impact of Striga, these are not effective on their own and require high monetary investments by smallholder farmers. My PhD research focuses on the potential of soil microbes to disrupt the early stages of the parasite’s life cycle through the production of volatile organic compounds. More specifically, we developed a computer vision tool that enabled the large-scale screening of a large bacterial collection for its functional potential to suppress Striga seed germination by naturally produced volatile compounds. This was complemented with the identification of several Striga-suppressive volatile compounds and studies into their genomic regulation. We developed a new approach of ‘precursor-directed activation’ of Striga-suppressive soil microbes by amending field soils with amino acid precursors to suppressive volatile compounds. This strategy will enable better deployment of volatile-mediated Striga suppression under field settings, by steering its production in situ and by aiding in the development of future control methods with higher efficacies and lower application costs Show less
Streptomycetes are soil-dwelling Gram-positive bacteria and can produce clinical drugs and adapt to environmental fluctuations through diverse morphological changes. The morphological development... Show moreStreptomycetes are soil-dwelling Gram-positive bacteria and can produce clinical drugs and adapt to environmental fluctuations through diverse morphological changes. The morphological development of streptomycetes features apical growth, which is determined by a tip-organizing center containing machinery for the synthesis of a cellulose-like glycan deposited at the cell surface. This thesis demonstrated that the cellulose-like glycan likely cellulose and the related synthesis machinery are unique in comparison with other Gram-negative cellulose synthesis systems. It showed that the synthesis of this glycan is performed by the glycosyltransferase ClsA and matured galactose-oxidase protein GlxA. Subsequently, the lytic polysaccharides monooxygenase LpmP and the glucanase ClsZ, adjacent to CslA/GlxA, cooperatively cleave peptidoglycan and create a passage to assist the exposure of this glycan on cell surface. In addition, this thesis also revealed coordination of polar growth determinants is performed by a stomatin-like protein StlP, which locates in the same gene cluster with CslA/GlxA/CslZ/LpmP. Oligomerization of StlP forms a fluid microdomain at hyphal tips, which spatially confines the whole tip organizing center. Furthermore, this thesis further showed that the cellulose-like glycan of Streptomyces coelicolor is responsible for the aggregation of clumps, which is distinct from Streptomyces lividans where this glycan mediates germlings’ aggregation. Show less
Calamus javensis is polymorphic. Morphometry and molecular approach resulted in all names used in the complex are regarded as synonyms of C. javensis except C. tenompokensis, which is retained as a... Show moreCalamus javensis is polymorphic. Morphometry and molecular approach resulted in all names used in the complex are regarded as synonyms of C. javensis except C. tenompokensis, which is retained as a distinct species. One variety is here recognized, C. javensis var. polyphyllus (including C. acuminatus) Show less
Sound is everywhere in the marine environment and hearing is therefore a very important for stimuli for fish. They use sound for orientation and communication, during migration, aggregation and... Show moreSound is everywhere in the marine environment and hearing is therefore a very important for stimuli for fish. They use sound for orientation and communication, during migration, aggregation and spawning, but also for detection of prey and predators. Understanding how fish are effected by sound has become increasingly important in light of the increasing contribution of noise produced by human activity at sea. In this thesis, I investigated the effects of two anthropogenic sound sources in relation to the movement behaviour of free-swimming Atlantic cod (Gadus morhua) in the North Sea: seismic surveying for the exploration of the seabed and pile driving during the construction of offshore windfarms. Results from the two sound exposure studies demonstrated that both seismic and piling sound effect the behaviour of cod. Exposure to a seismic survey resulted in reduced activity during exposure and fish leaving the area earlier in the year. Piling did not result in leaving but did cause cod to move closer to the hard bottom structures. Ultimately, these insights will bring us one step closer towards recognising the effects of anthropogenic noise on the behaviour of individual marine fish and the potential consequences this may have at population level. Show less
Streptomyces bacteria are a valuable source of natural products, many of which are used in the clinic or in biotechnology. In our search for novel antibiotics we discovered lugdunomycin, a natural... Show moreStreptomyces bacteria are a valuable source of natural products, many of which are used in the clinic or in biotechnology. In our search for novel antibiotics we discovered lugdunomycin, a natural product with a highly complex chemical architecture that is produced by Streptomyces sp. QL37. It is derived from the angucyclines, a well-known class of molecules known for their antibacterial and anticancer activities. Though angucyclines are produced in high quantities under most conditions, lugdunomycin is produced in minimal amounts. This thesis describes novel insights into the transcriptional control of the lugdunomycin biosynthetic gene cluster and into the lugdunomycin biosynthesis pathway. These insights may be applied to improve the yield of lugdunomycin and expand the chemical diversity of angucyclines. Using molecular biology, bioinformatic approaches and omics studies, such as metabolomics and transcriptomics, we have characterized the lugdunomycin biosynthetic gene cluster, the regulatory genes (lugRI–lugRV) required for transcriptional activation of the cluster, and the oxygenase genes (lugOI–lugOV) that play a key role in the different chemical rearrangements of the angucyclines. Furthermore, this thesis contains a detailed review of the regulatory network that controls antibiotic production in Actinobacteria. Show less
The largest genus within the Phyllanthaceae family is a group called Phyllanthus L. Recent studies have shown, that Phyllanthus is paraphyletic with the genera Glochidion, Breynia and Synostemon... Show moreThe largest genus within the Phyllanthaceae family is a group called Phyllanthus L. Recent studies have shown, that Phyllanthus is paraphyletic with the genera Glochidion, Breynia and Synostemon nested within it. In this thesis, I study the evolution and systematics of Phyllanthus and how to solve the apparent paraphyly. First we explore past morphological subgroups to come to a subdivision, which was tested using molecular phylogenetics. Several species of Phyllanthus are also used in traditional medicine and by utilizing the living collections of the Hortus botanicus Leiden, we tested for antibacterial and antifungal bioactivity. The phylogeny resulting from this study is used to study the historical biogeography and diversification of the tribe and to propose a new classification where Phyllanthus is separated into several smaller genera. Show less
Single-Molecule Microscopy (SMM) techniques constitute a group of powerful imaging tools that enable researchers to study the dynamic behavior of individual molecules.In the research described in... Show moreSingle-Molecule Microscopy (SMM) techniques constitute a group of powerful imaging tools that enable researchers to study the dynamic behavior of individual molecules.In the research described in this doctoral thesis, SMM techniques have been developed to image individual proteins inside cells of a living zebrafish embryo model and to study patterns of their mobility.The results of the mobility pattern analyses offer new insights into the dynamics of single molecules diffusing inside cells within the context of an intact vertebrate organism. Show less
Humans and songbirds learn their vocalizations early in life by exposure to the vocalizations of adult conspecifics. Often, better learning outcomes are achieved with live, social, tutors than with... Show moreHumans and songbirds learn their vocalizations early in life by exposure to the vocalizations of adult conspecifics. Often, better learning outcomes are achieved with live, social, tutors than with audio-only exposure to vocalizations. Many researchers argue that this is because social tutor-tutee interactions are important in the vocal learning process. It is unclear, however, whether and to what extent live tutoring also facilitates song learning because it results in multimodal exposure to a tutor, as tutees can both see and hear their tutor, while audio-only tutoring results in unimodal exposure. This thesis investigates whether multi- compared to unimodal exposure affects song learning in zebra finches, the primary experimental animal model for vocal learning studies, by revisiting the zebra finch song learning literature and describing three song tutoring experiments. In these experiments, young birds were provided with audio-only or audio-visual tutor exposure, using one-way mirrors, videos and robotic birds. The results suggest that multi- versus unimodal exposure to a live tutor can affect the timing of vocal development and possibly the amount of vocal learning. Multimodal exposure to artificial tutors made stimulus presentation more salient but did not affect the song learning outcomes assessed in this thesis. Show less
Filamentous Actinobacteria, such as Streptomyces, produce a plethora of chemically diverse bioactive metabolites that have found applications across medicine, agriculture and biotechnology. Yet,... Show moreFilamentous Actinobacteria, such as Streptomyces, produce a plethora of chemically diverse bioactive metabolites that have found applications across medicine, agriculture and biotechnology. Yet, the vast majority of the biosynthetic potential of Actinobacteria remains uncharacterised, largely because their biosynthetic gene clusters (BGCs) are poorly expressed in the laboratory, preventing the discovery of the cognate natural products. Additionally, only a narrow band of environments and a few taxonomic groups have been explored for gifted Actinobacteria. In this thesis different approaches are described, wherein we combined drug discovery with ecology, aimed at accessing the full potential of Actinobacteria. Bioactive Actinobacteria were isolated from a faecal sample of a 28,000-year-old-mammoth and their taxonomic and metabolic diversity was analysed. Furthermore, the effect of human stress hormones on the production of antibiotics by Streptomyces was investigated, resulting in the discovery of adrenaline as elicitor of siderophore production. This was later shown to be caused by the adrenaline analog catechol, which is ubiquitous in nature. Catechol also elicited the production of angucycline glycosides, well known for their therapeutic potential as anticancer and antibiotic compounds. Lastly, zebrafish were used as an in vivo model to explore the bioactive and functional potential of Actinobacteria within the animal microbiome. Show less
Vancomycin is a last-resort antibiotic for the treatment of many Gram-positive bacterial infections, while remaining inactive against Gram-negative strains. Resistance to vancomycin in Gram... Show moreVancomycin is a last-resort antibiotic for the treatment of many Gram-positive bacterial infections, while remaining inactive against Gram-negative strains. Resistance to vancomycin in Gram-positive stains continues to develop. This thesis describes the recent developments in semisynthetically modifying glycopeptide antibiotics to improve their antibacterial activity. Furthermore, the development of several semisynthetic glycopeptide antibiotics are described including the guanidino lipoglycopeptides, the vancomyxins, and the vancomycin-sideromycins. The guanidino lipoglycopeptides are readily synthesized from vancomycin and display potent in vitro and in vivo activity against Gram-positive bacteria, including vancomycin-resistant strains. Assessment of the activity, properties, and mechanism of action of the guanidino lipoglycopeptides shows the potential of these novel glycopeptides to become best-in class. The vancomyxins, which consist of covalently conjugated vancomycin and outer membrane disruptor polymyxin nonapeptide, display enhanced activity against Gram-negative bacterial strains compared to vancomycin monotherapy or co-administration of the two components. The vancomycin-sideromycins are also aimed at conferring antibacterial activity against Gram-negative bacteria by exploiting an iron-uptake system. Overall, a variety of semisynthetic vancomycin derivatives, aimed at overcoming vancomycin resistance or sensitizing Gram-negative strains, are developed and assessed on their activity in this work. Show less
Extending our current arsenal of antibiotics is key to staying ahead in the arms race between humans and resistant bacteria. Classes of antibiotics otherwise limited to the treatment of Gram... Show moreExtending our current arsenal of antibiotics is key to staying ahead in the arms race between humans and resistant bacteria. Classes of antibiotics otherwise limited to the treatment of Gram-positive pathogens may be potentiated against Gram-negative bacteria by disruption of their outer membrane. The work described in this thesis focuses on the development of novel synergists designed to selectively disrupt the outer membrane and in doing potentiate the activity of antibiotics that are otherwise inactive against Gram-negative bacteria. Show less
