Wired to the roots: Impact of root-beneficial microbe interactions on aboveground plant physiology and protection

Often, plant-pathogenic microbe interactions are discussed in a host-microbe two-component system, however very little is known about how the diversity of rhizospheric microbes that associate with plants affect host performance against pathogens. There are various studies, which specially direct the importance of induced systemic defense (ISR) response in plants interacting with beneficial rhizobacteria, yet we don’t know how rhizobacterial associations modulate plant physiology. In here, we highlight the many dimensions within which plant roots associate with beneficial microbes by regulating aboveground physiology. We review approaches to study the causes and consequences of plant root association with beneficial microbes on aboveground plant-pathogen interactions. The review provides the foundations for future investigations into the impact of the root beneficial microbial associations on plant performance and innate defense responses.     

Genetic and molecular analysis of nematode-microbe interactions

Symbiosis, the living together of unlike organisms, such as between microbes and their multicellular eukaryotic hosts, can be categorized as parasitic, commensal or mutualistic. The establishment of symbiosis and the outcome of microbe-host interactions are dictated largely by both microbe- and host-derived factors. Over the last decade, the nematode Caenorhabditis elegans has provided a facile experimental system to study such interactions, with parasitic interactions being the primary focus. The myriad of genetic and molecular tools available has made C. elegans a powerful model system to interrogate the interactions between a host and its pathogens, and has provided a greater understanding of the molecular underpinnings of these interactions, many of which were found to be conserved across other taxa. Commensal and mutualistic interactions between worms and their microbes, although less studied, have the potential to enhance our understanding of genetic and molecular features underlying host-microbe interactions. Here, we highlight new insights obtained in delineating the signalling pathways that function within and between host cells in combating assaults from extracellular and intracellular pathogens. We also discuss potential new insights that could be gained from further studies into commensal and mutualistic relationships between nematodes and microbes.     

Identification of Specialists and Abundance-Occupancy Relationships among Intestinal Bacteria of Aves,Mammalia, and Actinopterygii

The coalescence of next-generation DNA sequencing methods, ecological perspectives, and bioinformatics analysis tools is rapidly advancing our understanding of the evolution and function of vertebrate-associated bacterial communities. Delineation of host-microbe associations has applied benefits ranging from clinical treatments to protecting our natural waters. Microbial communities follow some broad-scale patterns observed for macroorganisms, but it remains unclear how the specialization of intestinal vertebrate-associated communities to a particular host environment influences broad-scale patterns in microbial abundance and distribution. We analyzed the V6 region of 16S rRNA genes amplified from 106 fecal samples spanning Aves, Mammalia, and Actinopterygii (ray-finned fish). We investigated the interspecific abundance-occupancy relationship, where widespread taxa tend to be more abundant than narrowly distributed taxa, among operational taxonomic units (OTUs) within and among host species. In a separate analysis, we identified specialist OTUs that were highly abundant in a single host and rare in all other hosts by using a multinomial model without excluding undersampled OTUs a priori. We show that intestinal microbes in humans and other vertebrates display abundance-occupancy relationships, but because intestinal host-associated communities have undergone intense specialization, this trend is violated by a disproportionately large number of specialist taxa. Although it is difficult to distinguish the effects of dispersal limitations, host selection, historical contingency, and stochastic processes on community assembly, results suggest that intestinal bacteria can be shared among diverse hosts in ways that resemble the distribution of “free-living” bacteria in the extraintestinal environment.     

Local Modelling Techniques for Assessing Micro-Level Impacts of Risk Factors in Complex Data: Understanding Health and Socioeconomic Inequalities in Childhood Educational Attainments

Although inequalities in health and socioeconomic status have an important influence on childhood educational performance, the interactions between these multiple factors relating to variation in educational outcomes at micro-level is unknown, and how to evaluate the many possible interactions of these factors is not well established. This paper aims to examine multi-dimensional deprivation factors and their impact on childhood educational outcomes at micro-level, focusing on geographic areas having widely different disparity patterns, in which each area is characterised by six deprivation domains (Income, Health, Geographical Access to Services, Housing, Physical Environment, and Community Safety). Traditional health statistical studies tend to use one global model to describe the whole population for macro-analysis. In this paper, we combine linked educational and deprivation data across small areas (median population of 1500), then use a local modelling technique, the Takagi-Sugeno fuzzy system, to predict area educational outcomes at ages 7 and 11. We define two new metrics, “Micro-impact of Domain” and “Contribution of Domain”, to quantify the variations of local impacts of multidimensional factors on educational outcomes across small areas. The two metrics highlight differing priorities. Our study reveals complex multi-way interactions between the deprivation domains, which could not be provided by traditional health statistical methods based on single global model. We demonstrate that although Income has an expected central role, all domains contribute, and in some areas Health, Environment, Access to Services, Housing and Community Safety each could be the dominant factor. Thus the relative importance of health and socioeconomic factors varies considerably for different areas, depending on the levels of each of the other factors, and therefore each component of deprivation must be considered as part of a wider system. Childhood educational achievement could benefit from policies and intervention strategies that are tailored to the local geographic areas'' profiles.     

Host innate immune receptors and beyond: making sense of microbial infections

The complexity of the immune system mirrors its manifold mechanisms of host-microbe interactions. A relatively simplified view was posited after the identification of host innate immune receptors that their distinct mechanisms of sensing "microbial signatures" create unique molecular switches to trigger the immune system. Recently, more sophisticated and cooperative strategies for these receptors have been revealed during receptor-ligand interactions, trafficking, and intra- and intercellular signaling, in order to deal with a diverse range of microbes. Continued mapping of the complex networks of host-microbe interactions may improve our understanding of self/non-self discrimination in immunity and its intervention.     

Protein Interactions in Genome Maintenance as Novel Antibacterial Targets

Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction “hubs” that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.     

Identifying Keystone Species in the Human Gut Microbiome from Metagenomic Timeseries Using Sparse Linear Regression

Human associated microbial communities exert tremendous influence over human health and disease. With modern metagenomic sequencing methods it is now possible to follow the relative abundance of microbes in a community over time. These microbial communities exhibit rich ecological dynamics and an important goal of microbial ecology is to infer the ecological interactions between species directly from sequence data. Any algorithm for inferring ecological interactions must overcome three major obstacles: 1) a correlation between the abundances of two species does not imply that those species are interacting, 2) the sum constraint on the relative abundances obtained from metagenomic studies makes it difficult to infer the parameters in timeseries models, and 3) errors due to experimental uncertainty, or mis-assignment of sequencing reads into operational taxonomic units, bias inferences of species interactions due to a statistical problem called “errors-in-variables”. Here we introduce an approach, Learning Interactions from MIcrobial Time Series (LIMITS), that overcomes these obstacles. LIMITS uses sparse linear regression with boostrap aggregation to infer a discrete-time Lotka-Volterra model for microbial dynamics. We tested LIMITS on synthetic data and showed that it could reliably infer the topology of the inter-species ecological interactions. We then used LIMITS to characterize the species interactions in the gut microbiomes of two individuals and found that the interaction networks varied significantly between individuals. Furthermore, we found that the interaction networks of the two individuals are dominated by distinct “keystone species”, Bacteroides fragilis and Bacteroided stercosis, that have a disproportionate influence on the structure of the gut microbiome even though they are only found in moderate abundance. Based on our results, we hypothesize that the abundances of certain keystone species may be responsible for individuality in the human gut microbiome.     

A Boolean Model of the Pseudomonas syringae hrp Regulon Predicts a Tightly Regulated System

The Type III secretion system (TTSS) is a protein secretion machinery used by certain gram-negative bacterial pathogens of plants and animals to deliver effector molecules to the host and is at the core of the ability to cause disease. Extensive molecular and biochemical study has revealed the components and their interactions within this system but reductive approaches do not consider the dynamical properties of the system as a whole. In order to gain a better understanding of these dynamical behaviours and to create a basis for the refinement of the experimentally derived knowledge we created a Boolean model of the regulatory interactions within the hrp regulon of Pseudomonas syringae pathovar tomato strain DC3000 Pseudomonas syringae. We compared simulations of the model with experimental data and found them to be largely in accordance, though the hrpV node shows some differences in state changes to that expected. Our simulations also revealed interesting dynamical properties not previously predicted. The model predicts that the hrp regulon is a biologically stable two-state system, with each of the stable states being strongly attractive, a feature indicative of selection for a tightly regulated and responsive system. The model predicts that the state of the GacS/GacA node confers control, a prediction that is consistent with experimental observations that the protein has a role as master regulator. Simulated gene “knock out” experiments with the model predict that HrpL is a central information processing point within the network.     

Antimicrobials,Stress and Mutagenesis

Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes whereas antibiotics are mostly employed by microorganisms. As antimicrobial peptides (AMPs) mostly target the cell wall, a microbial ‘Achilles heel’, it has been proposed that bacterial resistance evolution is very unlikely and hence AMPs are ancient ‘weapons’ of multicellular organisms. Here we provide a new hypothesis to explain the widespread distribution of AMPs amongst multicellular organism. Studying five antimicrobial peptides from vertebrates and insects, we show, using a classic Luria-Delbrück fluctuation assay, that cationic antimicrobial peptides (AMPs) do not increase bacterial mutation rates. Moreover, using rtPCR and disc diffusion assays we find that AMPs do not elicit SOS or rpoS bacterial stress pathways. This is in contrast to the main classes of antibiotics that elevate mutagenesis via eliciting the SOS and rpoS pathways. The notion of the ‘Achilles heel’ has been challenged by experimental selection for AMP-resistance, but our findings offer a new perspective on the evolutionary success of AMPs. Employing AMPs seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs.     

Identification of Polyketide Inhibitors Targeting 3-Dehydroquinate Dehydratase in the Shikimate Pathway of Enterococcus faecalis

Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the “drug of last resort”. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.     

Where Next for Microbiome Research?

The development of high-throughput sequencing technologies has transformed our capacity to investigate the composition and dynamics of the microbial communities that populate diverse habitats. Over the past decade, these advances have yielded an avalanche of metagenomic data. The current stage of “van Leeuwenhoek”–like cataloguing, as well as functional analyses, will likely accelerate as DNA and RNA sequencing, plus protein and metabolic profiling capacities and computational tools, continue to improve. However, it is time to consider: what’s next for microbiome research? The short pieces included here briefly consider the challenges and opportunities awaiting microbiome research.

This Perspective is part of the “Where next?” Series.

Soon, we will enter an era when “the number of population genomes deposited in public databases will dwarf those from isolates and single cells” (Gene Tyson). Clearly, as all authors noted in the following, our focus will move from describing the composition of microbial communities to elucidating the principles that govern their assembly, dynamics, and functions. How will such principles be discovered? Elhanan Borenstein proposes that a systems biology–based approach, particularly the development of mathematical and computational models of the interactions between the specific community components, will be critical for understanding the function and dynamics of microbiomes. Evolutionary biologists Howard Ochman and Andrew Moeller want to decipher how microbial assemblies evolve but challenge us to also consider the role of microbial communities in organismal evolution, and they make the exciting prediction that microbes will be implicated in the evolution of eusociality and cooperation. Brett Finlay underscores the need for deciphering the mechanistic bases—particularly the chemical/metabolite signals—for interactions between members of microbial communities and their hosts. He emphasizes how this knowledge will enable creation of new tools to manipulate the microbiota, a key challenge for future investigation. Heidi Kong also encourages deciphering the mechanisms that underlie associations between particular skin surfaces and disorders and their respective microbiota. Jeffrey Gordon considers several intriguing opportunities as well as challenges that manipulation of the gut microbiota presents for improved human nutrition and health. Finally, Karen Nelson, Karim Dabbagh and Hamilton Smith suggest that using synthetic genomes to create novel microbes or even synthetic microbiomes offers a new way to engineer the microbiota. Overall, future microbiome research regarding the molecules and mechanisms mediating interactions between members of microbial communities and their hosts should lead to discovery of exciting new biology and transformative therapeutics.     

From decision to action: Detailed modelling of frog tadpoles reveals neuronal mechanisms of decision-making and reproduces unpredictable swimming movements in response to sensory signals

How does the brain process sensory stimuli, and decide whether to initiate locomotor behaviour? To investigate this question we develop two whole body computer models of a tadpole. The “Central Nervous System” (CNS) model uses evidence from whole-cell recording to define 2300 neurons in 12 classes to study how sensory signals from the skin initiate and stop swimming. In response to skin stimulation, it generates realistic sensory pathway spiking and shows how hindbrain sensory memory populations on each side can compete to initiate reticulospinal neuron firing and start swimming. The 3-D “Virtual Tadpole” (VT) biomechanical model with realistic muscle innervation, body flexion, body-water interaction, and movement is then used to evaluate if motor nerve outputs from the CNS model can produce swimming-like movements in a volume of “water”. We find that the whole tadpole VT model generates reliable and realistic swimming. Combining these two models opens new perspectives for experiments.     

Stochastic Resonance Crossovers in Complex Networks

Here we numerically study the emergence of stochastic resonance as a mild phenomenon and how this transforms into an amazing enhancement of the signal-to-noise ratio at several levels of a disturbing ambient noise. The setting is a cooperative, interacting complex system modelled as an Ising-Hopfield network in which the intensity of mutual interactions or “synapses” varies with time in such a way that it accounts for, e.g., a kind of fatigue reported to occur in the cortex. This induces nonequilibrium phase transitions whose rising comes associated to various mechanisms producing two types of resonance. The model thus clarifies the details of the signal transmission and the causes of correlation among noise and signal. We also describe short-time persistent memory states, and conclude on the limited relevance of the network wiring topology. Our results, in qualitative agreement with the observation of excellent transmission of weak signals in the brain when competing with both intrinsic and external noise, are expected to be of wide validity and may have technological application. We also present here a first contact between the model behavior and psychotechnical data.     

Molecular defense responses in roots and the rhizosphere against Fusarium oxysporum

Plants face many different concurrent and consecutive abiotic and biotic stresses during their lifetime. Roots can be infected by numerous pathogens and parasitic organisms. Unlike foliar pathogens, root pathogens have not been explored enough to fully understand root-pathogen interactions and the underlying mechanism of defense and resistance. PR gene expression, structural responses, secondary metabolite and root exudate production, as well as the recruitment of plant defense–assisting “soldier” rhizosphere microbes all assist in root defense against pathogens and herbivores. With new high-throughput molecular tools becoming available and more affordable, now is the opportune time to take a deep look below the ground. In this addendum, we focus on soil-borne Fusarium oxysporum as a pathogen and the options plants have to defend themselves against these hard-to-control pathogens.     

Virulence or Niche Factors: What's in a Name?

The increasing interest in the human microbiota raises some interesting questions about the terminology we use to describe some of the structures and strategies employed by commensal and pathogenic microbes to compete in these complex biological ecosystems. For example, all microbes arriving in the alimentary tract face the task of surviving passage through the stomach, coping with bile, interacting with the immune system, competing with the established microbiota, and obtaining sufficient nutrients to gain a foothold in this hostile environment. It is not surprising then that many gastrointestinal microbes (both pathogens and commensals) use similar strategies to overcome the challenges associated with this particular biological niche. Given that many of these structures and strategies were discovered and characterized in pathogens and because they often play important roles in establishing and maintaining an infection, they have often been characterized as virulence factors. It would be misleading to describe the same strategies and structures found in harmless commensals as “virulence factors,” since they represent a sine qua non for life in the gastrointestinal tract. It may be time to reconsider and refer to them as “niche factors,” both in terms of providing scientific accuracy but also in light of the growing interest in using gut microbes as probiotics, where the distinction between virulence factors and niche factors is likely to be very important from a regulatory perspective.     

Natural experiments and long-term monitoring are critical to understand and predict marine host–microbe ecology and evolution

Marine multicellular organisms host a diverse collection of bacteria, archaea, microbial eukaryotes, and viruses that form their microbiome. Such host-associated microbes can significantly influence the host’s physiological capacities; however, the identity and functional role(s) of key members of the microbiome (“core microbiome”) in most marine hosts coexisting in natural settings remain obscure. Also unclear is how dynamic interactions between hosts and the immense standing pool of microbial genetic variation will affect marine ecosystems’ capacity to adjust to environmental changes. Here, we argue that significantly advancing our understanding of how host-associated microbes shape marine hosts’ plastic and adaptive responses to environmental change requires (i) recognizing that individual host–microbe systems do not exist in an ecological or evolutionary vacuum and (ii) expanding the field toward long-term, multidisciplinary research on entire communities of hosts and microbes. Natural experiments, such as time-calibrated geological events associated with well-characterized environmental gradients, provide unique ecological and evolutionary contexts to address this challenge. We focus here particularly on mutualistic interactions between hosts and microbes, but note that many of the same lessons and approaches would apply to other types of interactions.

This Essay argues that in order to truly understand how marine hosts benefit from the immense diversity of microbes, we need to expand towards long-term, multi-disciplinary research focussing on few areas of the world’s ocean that we refer to as “natural experiments,” where processes can be studied at scales that far exceed those captured in laboratory experiments.     

Aphid-Symbiotic Bacteria Cultured in Insect Cell Lines

The cells and tissues of many aphids contain bacteria known as “secondary symbionts,” which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names “Candidatus Consessoris aphidicola” for T type and “Candidatus Adiaceo aphidicola” for U type.     

Antimicrobial Functions of Lactoferrin Promote Genetic Conflicts in Ancient Primates and Modern Humans

Lactoferrin is a multifunctional mammalian immunity protein that limits microbial growth through sequestration of nutrient iron. Additionally, lactoferrin possesses cationic protein domains that directly bind and inhibit diverse microbes. The implications for these dual functions on lactoferrin evolution and genetic conflicts with microbes remain unclear. Here we show that lactoferrin has been subject to recurrent episodes of positive selection during primate divergence predominately at antimicrobial peptide surfaces consistent with long-term antagonism by bacteria. An abundant lactoferrin polymorphism in human populations and Neanderthals also exhibits signatures of positive selection across primates, linking ancient host-microbe conflicts to modern human genetic variation. Rapidly evolving sites in lactoferrin further correspond to molecular interfaces with opportunistic bacterial pathogens causing meningitis, pneumonia, and sepsis. Because microbes actively target lactoferrin to acquire iron, we propose that the emergence of antimicrobial activity provided a pivotal mechanism of adaptation sparking evolutionary conflicts via acquisition of new protein functions.     

Constraints on the deformation of the vibrissa within the follicle

Nearly all mammals have a vibrissal system specialized for tactile sensation, composed of whiskers growing from sensor-rich follicles in the skin. When a whisker deflects against an object, it deforms within the follicle and exerts forces on the mechanoreceptors inside. In addition, during active whisking behavior, muscle contractions around the follicle and increases in blood pressure in the ring sinus will affect the whisker deformation profile. To date, however, it is not yet possible to experimentally measure how the whisker deforms in an intact follicle or its effects on different groups of mechanoreceptors. The present study develops a novel model to predict vibrissal deformation within the follicle sinus complex. The model is based on experimental results from a previous ex vivo study on whisker deformation within the follicle, and on a new histological analysis of follicle tissue. It is then used to simulate whisker deformation within the follicle during passive touch and active whisking. Results suggest that the most likely whisker deformation profile is “S-shaped,” crossing the midline of the follicle right below the ring sinus. Simulations of active whisking indicate that an increase in overall muscle stiffness, an increase in the ratio between deep and superficial intrinsic muscle stiffness, and an increase in sinus blood pressure will all enhance tactile sensitivity. Finally, we discuss how the deformation profiles might map to the responses of primary afferents of each mechanoreceptor type. The mechanical model presented in this study is an important first step in simulating mechanical interactions within whisker follicles.