Towards engineering ectomycorrhization into switchgrass bioenergy crops via a lectin receptor-like kinase.

Soil-borne microbes can establish compatible relationships with host plants, providing a large variety of nutritive and protective compounds in exchange for photosynthesized sugars. However, the molecular mechanisms mediating the establishment of these beneficial relationships remain unclear. Our previous genetic mapping and whole-genome resequencing studies identified a gene deletion event of a Populus trichocarpa lectin receptor-like kinase gene PtLecRLK1 in Populus deltoides that was associated with poor-root colonization by the ectomycorrhizal fungus Laccaria bicolor. By introducing PtLecRLK1 into a perennial grass known to be a non-host of L. bicolor, switchgrass (Panicum virgatum L.), we found that L. bicolor colonizes ZmUbipro-PtLecRLK1 transgenic switchgrass roots, which illustrates that the introduction of PtLecRLK1 has the potential to convert a non-host to a host of L. bicolor. Furthermore, transcriptomic and proteomic analyses on inoculated-transgenic switchgrass roots revealed genes/proteins overrepresented in the compatible interaction and underrepresented in the pathogenic defence pathway, consistent with the view that pathogenic defence response is down-regulated during compatible interaction. Metabolomic profiling revealed that root colonization in the transgenic switchgrass was associated with an increase in N-containing metabolites and a decrease in organic acids, sugars, and aromatic hydroxycinnamate conjugates, which are often seen in the early steps of establishing compatible interactions. These studies illustrate that PtLecRLK1 is able to render a plant susceptible to colonization by the ectomycorrhizal fungus L. bicolor and shed light on engineering mycorrhizal symbiosis into a non-host to enhance plant productivity and fitness on marginal lands.

Biosynthesis and characterization of deuterated chitosan in filamentous fungus and yeast.

Deuterated chitosan was produced from the filamentous fungus Rhizopus oryzae, cultivated with deuterated glucose in H2O medium, without the need for conventional chemical deacetylation. After extraction and purification, the chemical composition and structure were determined by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and small-angle neutron scattering (SANS). 13C NMR experiments provided additional information about the position of the deuterons in the glucoseamine backbone. The NMR spectra indicated that the deuterium incorporation at the non-exchangeable hydrogen positions of the aminoglucopyranosyl ring in the C3 - C5 positions was at least 60-80 %. However, the C2 position was deuterated at a much lower level (6%). Also, SANS showed that the structure of deuterated chitosan was very similar compared to the non-deuterated counterpart. The most abundant radii of the protiated and deuterated chitosan fibers were 54 Å and 60 Å, respectively, but there is a broader distribution of fiber radii in the protiated chitosan sample. The highly deuterated, soluble fungal chitosan described here can be used as a model material for studying chitosan-enzyme complexes for future neutron scattering studies. Because the physical behavior of non-deuterated fungal chitosan mimicked that of shrimp shell chitosan, the methods presented here represent a new approach to producing a high quality deuterated non-animal-derived aminopolysaccharide for studying the structure-function association of biocomposite materials in drug delivery, tissue engineering and other bioactive chitosan-based composites.

Phylogenetic diversity of 200+ isolates of the ectomycorrhizal fungus Cenococcum geophilum associated with Populus trichocarpa soils in the Pacific Northwest, USA and comparison to globally distributed representatives.

The ectomycorrhizal fungal symbiont Cenococcum geophilum is of high interest as it is globally distributed, associates with many plant species, and has resistance to multiple environmental stressors. C. geophilum is only known from asexual states but is often considered a cryptic species complex, since extreme phylogenetic divergence is often observed within nearly morphologically identical strains. Alternatively, C. geophilum may represent a highly diverse single species, which would suggest cryptic but frequent recombination. Here we describe a new isolate collection of 229 C. geophilum isolates from soils under Populus trichocarpa at 123 collection sites spanning a ~283 mile north-south transect in Western Washington and Oregon, USA (PNW). To further understanding of the phylogenetic relationships within C. geophilum, we performed maximum likelihood and Bayesian phylogenetic analyses to assess divergence within the PNW isolate collection, as well as a global phylogenetic analysis of 789 isolates with publicly available data from the United States, Japan, and European countries. Phylogenetic analyses of the PNW isolates revealed three distinct phylogenetic groups, with 15 clades that strongly resolved at >80% bootstrap support based on a GAPDH phylogeny and one clade segregating strongly in two principle component analyses. The abundance and representation of PNW isolate clades varied greatly across the North-South range, including a monophyletic group of isolates that spanned nearly the entire gradient at ~250 miles. A direct comparison between the GAPDH and ITS rRNA gene region phylogenies, combined with additional analyses revealed stark incongruence between the ITS and GAPDH gene regions, consistent with intra-species recombination between PNW isolates. In the global isolate collection phylogeny, 34 clades were strongly resolved using Maximum Likelihood and Bayesian approaches (at >80% MLBS and >0.90 BPP respectively), with some clades having intra- and intercontinental distributions. Together these data are highly suggestive of divergence within multiple cryptic species, however additional analyses such as higher resolution genotype-by-sequencing approaches are needed to distinguish potential species boundaries and the mode and tempo of recombination patterns.

amPEPpy 1.0: a portable and accurate antimicrobial peptide prediction tool.

SUMMARY: Antimicrobial peptides (AMPs) are promising alternative antimicrobial agents. Currently, however, portable, user-friendly and efficient methods for predicting AMP sequences from genome-scale data are not readily available. Here we present amPEPpy, an open-source, multi-threaded command-line application for predicting AMP sequences using a random forest classifier. AVAILABILITY AND IMPLEMENTATION: amPEPpy is implemented in Python 3 and is freely available through GitHub (https://github.com/tlawrence3/amPEPpy). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Lipo-chitooligosaccharides as regulatory signals of fungal growth and development.

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.

Arabidopsis C-terminal binding protein ANGUSTIFOLIA modulates transcriptional co-regulation of MYB46 and WRKY33.

The apparent antagonism between salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signalling resulting in trade-offs between defence against (hemi)biotrophic and necrotrophic pathogens has been widely described across multiple plant species. However, the underlying mechanism remains to be fully established. The molecular and cellular functions of ANGUSTIFOLIA (AN) were characterised, and its role in regulating the pathogenic response was studied in Arabidopsis. We demonstrated that AN, a plant homologue of mammalian C-TERMINAL BINDING PROTEIN (CtBP), antagonistically regulates plant resistance to the hemibiotrophic pathogen Pseudomonas syringae and the necrotrophic pathogen Botrytis cinerea. Consistent with phenotypic observations, transcription of genes involved in SA and JA/ET pathways was antagonistically regulated by AN. By interacting with another nuclear protein TYROSYL-DNA PHOSPHODIESTERASE1 (TDP1), AN imposes transcriptional repression on MYB46, encoding a transcriptional activator of PHENYLALANINE AMMONIA-LYASE (PAL) genes which are required for SA biosynthesis, while releasing TDP1-imposed transcriptional repression on WRKY33, a master regulator of the JA/ET signalling pathway. These findings demonstrate that transcriptional co-regulation of MYB46 and WRKY33 by AN mediates the coordination of SA and JA/ET pathways to optimise defences against (hemi)biotrophic and necrotrophic pathogens.

Insight into a highly polymorphic endophyte isolated from the roots of the halophytic seepweed Suaeda salsa: Laburnicola rhizohalophila sp. nov. (Didymosphaeriaceae, Pleosporales).

We surveyed root endophytic fungi of the coastal halophyte Suaeda salsa and detected a population of a novel species that we described here as Laburnicola rhizohalophila sp. nov. No sexual sporulating structure was observed. Instead, it produced a large amount of thalloconidia, 0-1 transverse septa, hyaline to darkly pigmented, often peanut-shaped and sometimes dumbbell-shaped, both ends enlarged with numerous oil droplets inside the hyphal cells. Surprisingly, a high degree of phenotypic and physiological intraspecific variation (e.g., salinity tolerance, growth under different carbon:nitrogen ratios, and carbon utilization pattern) was recorded. The inoculation test indicated that the isolates could successfully infect host roots and form microsclerotia-like structures in cortical cells, a typical trait of dark septate endophytes (DSEs). Furthermore, most isolates were shown to promote host seedling growth. To evaluate conspecificity and infer its phylogenetic affinity, multiloci data including nuclear rRNA loci (ITS1 and 2, partial 28S), partial RNA Polymerase II second-largest subunit (rpb2), and partial translation elongation factor-1α (tef1) were characterized. Genealogical concordance phylogenetic species recognition (GCPSR) detected a genetically isolated clade of L. rhizohalophila within the Pleosporales in the Didymosphaeriaceae. Maximum likelihood phylogenetic reconstruction revealed that the endophytic fungus was genetically close to Laburnicoladactylidis but separated by a relatively long genetic distance. Our work highlights that the pleosporalean taxa might represent an underexplored reservoir of root DSEs.

Microbe to Microbiome: A Paradigm Shift in the Application of Microorganisms for Sustainable Agriculture.

Light, water and healthy soil are three essential natural resources required for agricultural productivity. Industrialization of agriculture has resulted in intensification of cropping practices using enormous amounts of chemical pesticides and fertilizers that damage these natural resources. Therefore, there is a need to embrace agriculture practices that do not depend on greater use of fertilizers and water to meet the growing demand of global food requirements. Plants and soil harbor millions of microorganisms, which collectively form a microbial community known as the microbiome. An effective microbiome can offer benefits to its host, including plant growth promotion, nutrient use efficiency, and control of pests and phytopathogens. Therefore, there is an immediate need to bring functional potential of plant-associated microbiome and its innovation into crop production. In addition to that, new scientific methodologies that can track the nutrient flux through the plant, its resident microbiome and surrounding soil, will offer new opportunities for the design of more efficient microbial consortia design. It is now increasingly acknowledged that the diversity of a microbial inoculum is as important as its plant growth promoting ability. Not surprisingly, outcomes from such plant and soil microbiome studies have resulted in a paradigm shift away from single, specific soil microbes to a more holistic microbiome approach for enhancing crop productivity and the restoration of soil health. Herein, we have reviewed this paradigm shift and discussed various aspects of benign microbiome-based approaches for sustainable agriculture.

Advancing How We Learn from Biodesign to Mitigate Risks with Next-Generation Genome Engineering.

In the last decade, the unprecedented simplicity and flexibility of the CRISPR-Cas system has made it the dominant transformative tool in gene and genome editing. However, this democratized technology is both a boon and a bane, for which we have yet to understand the full potential to investigate and rewrite genomes (also named "genome biodesign"). Rapid CRISPR advances in a range of applications in basic research, agriculture, and clinical applications pose new risks and raise several biosecurity concerns. In such a fast-moving field of research, we emphasize the importance of properly communicating the quality and accuracy of results and recommend new reporting requirements for results derived from next-generation genome engineering.

Plant Biosystems Design Research Roadmap 1.0.

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance.

Microfluidics and Metabolomics Reveal Symbiotic Bacterial-Fungal Interactions Between Mortierella elongata and Burkholderia Include Metabolite Exchange.

We identified two poplar (Populus sp.)-associated microbes, the fungus, Mortierella elongata strain AG77, and the bacterium, Burkholderia strain BT03, that mutually promote each other's growth. Using culture assays in concert with a novel microfluidic device to generate time-lapse videos, we found growth specific media differing in pH and pre-conditioned by microbial growth led to increased fungal and bacterial growth rates. Coupling microfluidics and comparative metabolomics data results indicated that observed microbial growth stimulation involves metabolic exchange during two ordered events. The first is an emission of fungal metabolites, including organic acids used or modified by bacteria. A second signal of unknown nature is produced by bacteria which increases fungal growth rates. We find this symbiosis is initiated in part by metabolic exchange involving fungal organic acids.

The Ectomycorrhizal Fungus Laccaria bicolor Produces Lipochitooligosaccharides and Uses the Common Symbiosis Pathway to Colonize Populus Roots.

Mycorrhizal fungi form mutualistic associations with the roots of most land plants and provide them with mineral nutrients from the soil in exchange for fixed carbon derived from photosynthesis. The common symbiosis pathway (CSP) is a conserved molecular signaling pathway in all plants capable of associating with arbuscular mycorrhizal fungi. It is required not only for arbuscular mycorrhizal symbiosis but also for rhizobia-legume and actinorhizal symbioses. Given its role in such diverse symbiotic associations, we hypothesized that the CSP also plays a role in ectomycorrhizal associations. We showed that the ectomycorrhizal fungus Laccaria bicolor produces an array of lipochitooligosaccharides (LCOs) that can trigger both root hair branching in legumes and, most importantly, calcium spiking in the host plant Populus in a CASTOR/POLLUX-dependent manner. Nonsulfated LCOs enhanced lateral root development in Populus in a calcium/calmodulin-dependent protein kinase (CCaMK)-dependent manner, and sulfated LCOs enhanced the colonization of Populus by L. bicolor Compared with the wild-type Populus, the colonization of CASTOR/POLLUX and CCaMK RNA interference lines by L. bicolor was reduced. Our work demonstrates that similar to other root symbioses, L. bicolor uses the CSP for the full establishment of its mutualistic association with Populus.

Mediation of plant-mycorrhizal interaction by a lectin receptor-like kinase.

The molecular mechanisms underlying mycorrhizal symbioses, the most ubiquitous and impactful mutualistic plant-microbial interaction in nature, are largely unknown. Through genetic mapping, resequencing and molecular validation, we demonstrate that a G-type lectin receptor-like kinase (lecRLK) mediates the symbiotic interaction between Populus and the ectomycorrhizal fungus Laccaria bicolor. This finding uncovers an important molecular step in the establishment of symbiotic plant-fungal associations and provides a molecular target for engineering beneficial mycorrhizal relationships.

Terpene Synthase Genes Originated from Bacteria through Horizontal Gene Transfer Contribute to Terpenoid Diversity in Fungi.

Fungi are successful eukaryotes of wide distribution. They are known as rich producers of secondary metabolites, especially terpenoids, which are important for fungi-environment interactions. Horizontal gene transfer (HGT) is an important mechanism contributing to genetic innovation of fungi. However, it remains unclear whether HGT has played a role in creating the enormous chemical diversity of fungal terpenoids. Here we report that fungi have acquired terpene synthase genes (TPSs), which encode pivotal enzymes for terpenoid biosynthesis, from bacteria through HGT. Phylogenetic analysis placed the majority of fungal and bacterial TPS genes from diverse taxa into two clades, indicating ancient divergence. Nested in the bacterial TPS clade is a number of fungal TPS genes that are inferred as the outcome of HGT. These include a monophyletic clade of nine fungal TPS genes, designated as BTPSL for bacterial TPS-like genes, from eight species of related entomopathogenic fungi, including seven TPSs from six species in the genus Metarhizium. In vitro enzyme assays demonstrate that all seven BTPSL genes from the genus Metarhizium encode active enzymes with sesquiterpene synthase activities of two general product profiles. By analyzing the catalytic activity of two resurrected ancestral BTPSLs and one closely related bacterial TPS, the trajectory of functional evolution of BTPSLs after HGT from bacteria to fungi and functional divergence within Metarhizium could be traced. Using M. brunneum as a model species, both BTPSLs and typical fungal TPSs were demonstrated to be involved in the in vivo production of terpenoids, illustrating the general importance of HGT of TPS genes from bacteria as a mechanism contributing to terpenoid diversity in fungi.

Increasing access to microfluidics for studying fungi and other branched biological structures.

BACKGROUND: Microfluidic systems are well-suited for studying mixed biological communities for improving industrial processes of fermentation, biofuel production, and pharmaceutical production. The results of which have the potential to resolve the underlying mechanisms of growth and transport in these complex branched living systems. Microfluidics provide controlled environments and improved optical access for real-time and high-resolution imaging studies that allow high-content and quantitative analyses. Studying growing branched structures and the dynamics of cellular interactions with both biotic and abiotic cues provides context for molecule production and genetic manipulations. To make progress in this arena, technical and logistical barriers must be overcome to more effectively deploy microfluidics in biological disciplines. A principle technical barrier is the process of assembling, sterilizing, and hydrating the microfluidic system; the lack of the necessary equipment for the preparatory process is a contributing factor to this barrier. To improve access to microfluidic systems, we present the development, characterization, and implementation of a microfluidics assembly and packaging process that builds on self-priming point-of-care principles to achieve "ready-to-use microfluidics." RESULTS: We present results from domestic and international collaborations using novel microfluidic architectures prepared with a unique packaging protocol. We implement this approach by focusing primarily on filamentous fungi; we also demonstrate the utility of this approach for collaborations on plants and neurons. In this work we (1) determine the shelf-life of ready-to-use microfluidics, (2) demonstrate biofilm-like colonization on fungi, (3) describe bacterial motility on fungal hyphae (fungal highway), (4) report material-dependent bacterial-fungal colonization, (5) demonstrate germination of vacuum-sealed Arabidopsis seeds in microfluidics stored for up to 2 weeks, and (6) observe bidirectional cytoplasmic streaming in fungi. CONCLUSIONS: This pre-packaging approach provides a simple, one step process to initiate microfluidics in any setting for fungal studies, bacteria-fungal interactions, and other biological inquiries. This process improves access to microfluidics for controlling biological microenvironments, and further enabling visual and quantitative analysis of fungal cultures.

Identification of Populus Small RNAs Responsive to Mutualistic Interactions With Mycorrhizal Fungi, Laccaria bicolor and Rhizophagus irregularis.

Ecto- and endo-mycorrhizal colonization of Populus roots have a positive impact on the overall tree health and growth. A complete molecular understanding of these interactions will have important implications for increasing agricultural or forestry sustainability using plant:microbe-based strategies. These beneficial associations entail extensive morphological changes orchestrated by the genetic reprogramming in both organisms. In this study, we performed a comparative analysis of two Populus species (Populus deltoides and P. trichocarpa) that were colonized by either an arbuscular mycorrhizal fungus (AmF), Rhizophagus irregularis or an ectomycorrhizal fungus (EmF), Laccaria bicolor, to describe the small RNA (sRNA) landscape including small open reading frames (sORFs) and micro RNAs (miRNAs) involved in these mutualistic interactions. We identified differential expression of sRNAs that were, to a large extent, (1) within the genomic regions lacking annotated genes in the Populus genome and (2) distinct for each fungal interaction. These sRNAs may be a source of novel sORFs within a genome, and in this regard, we identified potential sORFs encoded by the sRNAs. We predicted a higher number of differentially-expressed miRNAs in P. trichocarpa (4 times more) than in P. deltoides (conserved and novel). In addition, 44 miRNAs were common in P. trichocarpa between the EmF and AmF treatments, and only 4 miRNAs were common in P. deltoides between the treatments. Root colonization by either fungus was more effective in P. trichocarpa than in P. deltoides, thus the relatively few differentially-expressed miRNAs predicted in P. deltoides might reflect the extent of the symbiosis. Finally, we predicted several genes targets for the plant miRNAs identified here, including potential fungal gene targets. Our findings shed light on additional molecular tiers with a role in Populus-fungal mutualistic associations and provides a set of potential molecular targets for future enhancement.

Fungal Endophytes of Populus trichocarpa Alter Host Phenotype, Gene Expression, and Rhizobiome Composition.

Mortierella and Ilyonectria genera include common species of soil fungi that are frequently detected as root endophytes in many plants, including Populus spp. However, the ecological roles of these and other endophytic fungi with respect to plant growth and function are still not well understood. The functional ecology of two key taxa from the P. trichocarpa rhizobiome, M. elongata PMI93 and I. europaea PMI82, was studied by coupling forest soil bioassays with environmental metatranscriptomics. Using soil bioassay experiments amended with fungal inoculants, M. elongata was observed to promote the growth of P. trichocarpa. This response was cultivar independent. In contrast, I. europaea had no visible effect on P. trichocarpa growth. Metatranscriptomic studies revealed that these fungi impacted rhizophytic and endophytic activities in P. trichocarpa and induced shifts in soil and root microbial communities. Differential expression of core genes in P. trichocarpa roots was observed in response to both fungal species. Expression of P. trichocarpa genes for lipid signaling and nutrient uptake were upregulated, and expression of genes associated with gibberellin signaling were altered in plants inoculated with M. elongata, but not I. europaea. Upregulation of genes for growth promotion, downregulation of genes for several leucine-rich repeat receptor kinases, and alteration of expression of genes associated with plant defense responses (e.g., jasmonic acid, salicylic acid, and ethylene signal pathways) also suggest that M. elongata manipulates plant defenses while promoting plant growth.

Perspectives on the basic and applied aspects of crassulacean acid metabolism (CAM) research.

Due to public concerns about the decreasing supply of blue water and increasing heat and drought stress on plant growth caused by urbanization, increasing human population and climate change, interest in crassulacean acid metabolism (CAM), a specialized type of photosynthesis enhancing water-use efficiency (WUE) and drought tolerance, has increased markedly. Significant progress has been achieved in both basic and applied research in CAM plants since the beginning of this century. Here we provide a brief overview of the current status of CAM research, and discuss future needs and opportunities in a wide range of areas including systems biology, synthetic biology, and utilization of CAM crops for human benefit, with a focus on the following aspects: 1) application of genome-editing technology and high-throughput phenotyping to functional genomics research in model CAM species and genetic improvement of CAM crops, 2) challenges for multi-scale metabolic modeling of CAM systems, 3) opportunities and new strategies for CAM pathway engineering to enhance WUE and drought tolerance in C3 (and C4) photosynthesis crops, 4) potential of CAM species as resources for food, feed, natural products, pharmaceuticals and biofuels, and 5) development of CAM crops for ecological and aesthetic benefits.

Bacterial biofilm formation on the hyphae of ectomycorrhizal fungi: a widespread ability under controls?

Ectomycorrhizal (ECM) fungi establish symbiosis with roots of most trees of boreal and temperate ecosystems and are major drivers of nutrient fluxes between trees and the soil. ECM fungi constantly interact with bacteria all along their life cycle and the extended networks of hyphae provide a habitat for complex bacterial communities. Despite the important effects these bacteria can have on the growth and activities of ECM fungi, little is known about the mechanisms by which these microorganisms interact. Here we investigated the ability of bacteria to form biofilm on the hyphae of the ECM fungus Laccaria bicolor. We showed that the ability to form biofilms on the hyphae of the ECM fungus is widely shared among soil bacteria. Conversely, some fungi, belonging to the Ascomycete class, did not allow for the formation of bacterial biofilms on their surfaces. The formation of biofilms was also modulated by the presence of tree roots and ectomycorrhizae, suggesting that biofilm formation does not occur randomly in soil but that it is regulated by several biotic factors. In addition, our study demonstrated that the formation of bacterial biofilm on fungal hyphae relies on the production of networks of filaments made of extracellular DNA.

Bacterial-fungal interactions: ecology, mechanisms and challenges.

Fungi and bacteria are found living together in a wide variety of environments. Their interactions are significant drivers of many ecosystem functions and are important for the health of plants and animals. A large number of fungal and bacterial families engage in complex interactions that lead to critical behavioural shifts of the microorganisms ranging from mutualism to antagonism. The importance of bacterial-fungal interactions (BFI) in environmental science, medicine and biotechnology has led to the emergence of a dynamic and multidisciplinary research field that combines highly diverse approaches including molecular biology, genomics, geochemistry, chemical and microbial ecology, biophysics and ecological modelling. In this review, we discuss recent advances that underscore the roles of BFI across relevant habitats and ecosystems. A particular focus is placed on the understanding of BFI within complex microbial communities and in regard of the metaorganism concept. We also discuss recent discoveries that clarify the (molecular) mechanisms involved in bacterial-fungal relationships, and the contribution of new technologies to decipher generic principles of BFI in terms of physical associations and molecular dialogues. Finally, we discuss future directions for research in order to stimulate synergy within the BFI research area and to resolve outstanding questions.