Host genotype structures the microbiome of a globally dispersed marine phytoplankton

Phytoplankton support complex bacterial microbiomes that rely on phytoplankton-derived extracellular compounds and perform functions necessary for algal growth. Recent work has revealed sophisticated interactions and exchanges of molecules between specific phytoplankton–bacteria pairs, but the role of host genotype in regulating those interactions is unknown. Here, we show how phytoplankton microbiomes are shaped by intraspecific genetic variation in the host using global environmental isolates of the model phytoplankton host Thalassiosira rotula and a laboratory common garden experiment. A set of 81 environmental T. rotula genotypes from three ocean basins and eight genetically distinct populations did not reveal a core microbiome. While no single bacterial phylotype was shared across all genotypes, we found strong genotypic influence of T. rotula, with microbiomes associating more strongly with host genetic population than with environmental factors. The microbiome association with host genetic population persisted across different ocean basins, suggesting that microbiomes may be associated with host populations for decades. To isolate the impact of host genotype on microbiomes, a common garden experiment using eight genotypes from three distinct host populations again found that host genotype influenced microbial community composition, suggesting that a process we describe as genotypic filtering, analogous to environmental filtering, shapes phytoplankton microbiomes. In both the environmental and laboratory studies, microbiome variation between genotypes suggests that other factors influenced microbiome composition but did not swamp the dominant signal of host genetic background. The long-term association of microbiomes with specific host genotypes reveals a possible mechanism explaining the evolution and maintenance of complex phytoplankton–bacteria chemical exchanges.

Interactions between marine phytoplankton and bacteria can exert a profound influence on ecosystem function and biogeochemical cycling, impacting rates of primary production, phytoplankton aggregation, organic carbon export, and nutrient cycling (1–3). As an aquatic analog of the plant rhizosphere, the most intimate relationships between phytoplankton and bacteria exist in the phycosphere, the region immediately surrounding a phytoplankton cell, where molecules can be exchanged despite the effects of turbulence and diffusion (4). Relationships between phytoplankton and bacteria in the phycosphere range from cooperative to competitive (5). For example, during exponential growth, phytoplankton actively secrete amino acids that are taken up by bacteria, despite potentially significant energy costs (6). In turn, some bacteria synthesize essential vitamins and growth hormones that stimulate phytoplankton productivity (7, 8). During phytoplankton senescence, formerly “friendly” bacteria can become pathogenic, producing algicides that lyse phytoplankton cells and release organic carbon to the environment (9). These complex ecological interactions have been investigated in the laboratory for specific phytoplankton–bacteria pairs (3). However, the persistence and phylogenetic breadth of these relationships for both host and microbiome remain open questions (10–12).In terrestrial habitats, clear linkages exist between the genetic background (i.e., host genotype and population genetic structure) of foundational plant species and the organisms that rely on them. For example, genetic variation within tree species can influence the structure of associated epiphytic, mycorrhizal, and invertebrate communities (13–15) through mutualism, parasitism, commensalism, facilitation, and competition (reviewed in ref. 16). These associations extend to plant-associated bacteria, whose abundance, composition, and diversity reflect intraspecific trait variation among host genotypes (17). Because microbes regulate processes such as decomposition, nutrient dynamics, and energy flow, the influence of intraspecific genetic variation in plants on their associated bacteria extends the effects of community genetics to ecosystem processes (18). In contrast to terrestrial habitats, seawater allows both bacteria and their phytoplankton hosts to drift with nearly unlimited dispersal across the global ocean. It is unknown whether planktonic communities mirror those in terrestrial habitats, where host genetics can shape bacterial community composition and influence ecosystem processes (16), or whether dispersal in the dilute marine environment overwhelms the formation of close and specific relationships between phytoplankton and their associated microbiota. Although phytoplankton drift freely across the global ocean with nearly unlimited dispersal and divide primarily asexually, they still possess clear genetic structure. Phytoplankton species are organized into genetically distinct populations that possess phenotypic trait variation, are associated with specific environmental conditions, and undergo large increases in abundance, known as blooms (19–23). Furthermore, genetically distinct phytoplankton populations persist on time scales of decades to centuries (23, 24), providing ample opportunity for populations to develop specific relationships with other microbes. Few studies have evaluated the microbiomes of multiple strains within a phytoplankton species. Some studies found that individual phytoplankton species may possess a core microbiome, with a consistent set of bacterial phylotypes and metabolic potentials (25, 26), while others found that phytoplankton strains supported distinct microbiomes (10) and differed in their growth responses to the same bacterial strain (7, 27, 28). These intriguing findings have not been rigorously examined in light of the genetic background of the host phytoplankton species. Given that plant genotype often (29–31) but not always (32) drives host microbiomes and given the differences between terrestrial and planktonic habitats, understanding to what extent the genetic background of phytoplankton species influences microbiome composition is critical to understanding the nature of their interactions and parsing the roles of both partners in global biogeochemical cycles.Here, we assessed host–microbe interactions using the model marine phytoplankton Thalassiosira rotula, a cosmopolitan species characterized by high genotypic and phenotypic diversity, which is subdivided into genetically distinct populations (24, 33). We examined whether the T. rotula microbiome was influenced by host genetic background, either at the genotype or population level. We combined a study of the microbiomes of 81 environmental genotypes (representing eight genetically distinct populations) sampled from around the world (Fig. 1 and Open in a separate windowFig. 1.Global sampling locations of the phytoplankton host T. rotula populations [symbol colors denote populations identified in Whittaker and Rynearson (24), and black indicates populations identified in this study] and their associated microbiomes (SI Appendix, Table S1). Three sites (Wa, Nb, and Fr) were resampled (symbols with two colors), and whole seawater was collected twice from Narragansett Bay (Nb) for microbiome comparisons with the in situ whole seawater bacterial community (asterisks). The base map is a composite of log annual average chlorophyll a concentrations (milligrams/meter⁻³, 2010) (https://oceandata.sci.gsfc.nasa.gov/directaccess/MODIS-Aqua/Mapped/Annual/9km/chlor_a/).Table 1.Global sampling site information for T. rotula and associated bacteria

Microbiome reduction and endosymbiont gain from a switch in sea urchin life history

Animal gastrointestinal tracts harbor a microbiome that is integral to host function, yet species from diverse phyla have evolved a reduced digestive system or lost it completely. Whether such changes are associated with alterations in the diversity and/or abundance of the microbiome remains an untested hypothesis in evolutionary symbiosis. Here, using the life history transition from planktotrophy (feeding) to lecithotrophy (nonfeeding) in the sea urchin Heliocidaris, we demonstrate that the lack of a functional gut corresponds with a reduction in microbial community diversity and abundance as well as the association with a diet-specific microbiome. We also determine that the lecithotroph vertically transmits a Rickettsiales that may complement host nutrition through amino acid biosynthesis and influence host reproduction. Our results indicate that the evolutionary loss of a functional gut correlates with a reduction in the microbiome and the association with an endosymbiont. Symbiotic transitions can therefore accompany life history transitions in the evolution of developmental strategies.

Animal gastrointestinal tracts contain microbial communities that are integral to host metabolism, immunity, and development (1, 2). Symbioses between animals and their gut microbiome have deep evolutionary origins (1, 2), often exhibit phylosymbiosis (3), and can serve as a physiological buffer to heterogeneous environments (2). Despite the necessity of the gastrointestinal tract and benefits of the gut microbiome (3), species in various phyla have lost a functional digestive system (4, 5). Loss of a functional gut should, in theory, cascade into a reduction in microbial diversity and the loss of diet-induced shifts in microbiome composition. These nutritional shifts may then provide a niche for functionally important endosymbionts, such as the chemoautotrophic bacteria commonly associated with gutless invertebrates (6, 7).Major life history transitions are driven by tradeoffs in reproduction and development that, in turn, impact fitness (8). These tradeoffs are particularly evident in benthic marine invertebrates whose developmental stages broadly group into two alternative nutritional strategies (4, 9). The first—planktotrophy—typically includes the production of a high number of small, energy-poor eggs that develop into larvae with feeding structures used to collect and process exogenous resources required to reach metamorphic competency (4, 9). The second—lecithotrophy—involves the production of fewer large, energy-rich eggs and nonfeeding larvae that undergo metamorphosis without the requirement of external nutrients through feeding (4, 9). Life history transitions between these developmental modes have occurred in several major animal lineages, with rapid evolutionary shifts from planktotrophy to lecithotrophy being well documented in echinoderms (4, 5, 10–13). It is thought that an increase in the eggs energetic content relaxes the selective pressure maintaining the feeding structures (e.g., the larval arms and a functional gastrointestinal tract) and that development to metamorphosis is accelerated once these are lost (5).One of the most comprehensively studied systems for life history transitions among marine invertebrates involves species in the sea urchin genus Heliocidaris. A speciation event ∼5 Mya resulted in two sister species with alternative life history strategies: Heliocidaris tuberculata is planktotrophic while Heliocidaris erythrogramma is lecithotrophic (14). Typical of planktotrophs, H. tuberculata develops from small eggs into feeding larvae that exhibit morphological plasticity in response to food limitation (15), which is correlated with compositional shifts in the microbiome (16, 17). H. erythrogramma, on the other hand, develops from eggs ∼53× to 86× the volume of H. tuberculata (18), lacks the morphological structures required for feeding, and has a reduced, nonfunctional digestive tract (11). This life history switch and heterochronic shift in development (11) corresponds with a rewiring of the gene regulatory network (19), reorganization of cell fates (20), and modification to gametogenesis (21).Here, we compare the bacterial communities of these Heliocidaris species and test two hypotheses. First, we test whether the loss of gut function coincides with a reduction in microbial symbiont diversity, and second, by simulating the natural range in food availability, we also test that the loss in gut function coincides with a loss in diet-related shifts in the microbiome. We report major reductions in microbiome diversity and abundance as well as the absence of bacterial communities correlated with food availability for the lecithotrophic H. erythrogramma. Moreover, we find that this species vertically transmits a Rickettsiales that encodes pathways for the biosynthesis of essential amino acids, proteins with pivotal roles in host reproduction, and enzymes to metabolize diacylglycerol ethers, the major lipid group responsible for the increase in egg size in H. erythrogramma and that is used to fuel growth and development (18, 22).     

Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest

The phyllosphere—the aerial surfaces of plants, including leaves—is a ubiquitous global habitat that harbors diverse bacterial communities. Phyllosphere bacterial communities have the potential to influence plant biogeography and ecosystem function through their influence on the fitness and function of their hosts, but the host attributes that drive community assembly in the phyllosphere are poorly understood. In this study we used high-throughput sequencing to quantify bacterial community structure on the leaves of 57 tree species in a neotropical forest in Panama. We tested for relationships between bacterial communities on tree leaves and the functional traits, taxonomy, and phylogeny of their plant hosts. Bacterial communities on tropical tree leaves were diverse; leaves from individual trees were host to more than 400 bacterial taxa. Bacterial communities in the phyllosphere were dominated by a core microbiome of taxa including Actinobacteria, Alpha-, Beta-, and Gammaproteobacteria, and Sphingobacteria. Host attributes including plant taxonomic identity, phylogeny, growth and mortality rates, wood density, leaf mass per area, and leaf nitrogen and phosphorous concentrations were correlated with bacterial community structure on leaves. The relative abundances of several bacterial taxa were correlated with suites of host plant traits related to major axes of plant trait variation, including the leaf economics spectrum and the wood density–growth/mortality tradeoff. These correlations between phyllosphere bacterial diversity and host growth, mortality, and function suggest that incorporating information on plant–microbe associations will improve our ability to understand plant functional biogeography and the drivers of variation in plant and ecosystem function.The phyllosphere—the aerial surfaces of plants—is an important and ubiquitous habitat for bacteria (1). It is estimated that on a global scale, the phyllosphere spans more than 10⁸ km² and is home to up to 10²⁶ bacterial cells (2). Bacteria are also important to their plant hosts. Leaf-associated bacteria represent a widespread and ancient symbiosis (3, 4) that can influence host growth and function in many ways, including the production of growth-promoting nutrients and hormones (5, 6) and protection of hosts against pathogen infection (7, 8). Phyllosphere bacteria have the potential to influence plant biogeography and ecosystem function through their influence on plant performance under different environmental conditions (9–11), but the drivers of variation in leaf-associated bacterial biodiversity among host plants are not well understood.The ability to quantify microbial community structure in depth with environmental sequencing technologies has led to an increasing focus not only on the ecology of individual microbial taxa but on the entire genomic content of communities of microbes in different habitats, or “microbiomes” (12). Numerous studies of host-associated microbiomes have shown that microbial biodiversity is a trait (13) that forms part of the extended phenotype of the host organism (4, 14, 15) with important effects on the health and fitness (16–18) and evolution (19–21) of the host. Because of the importance of the microbiome for host fitness and function, there is a growing desire to model and manage host–microbiome interactions (22, 23), and understanding the drivers of host-associated microbial community assembly has thus become a cornerstone of microbiome research (24).In animals, the assembly of host-associated microbiomes is known to be driven by ecologically important host attributes, such as diet, that covary with host evolutionary history (20, 25, 26). A similar understanding of the drivers of plant microbiome assembly is lacking. Most of our knowledge of plant–bacterial associations on leaves has been based on studies of individual bacterial strains and individual host species. Different plant species possess characteristic bacterial phyllosphere communities (27, 28), and there are several examples of variation in bacterial biodiversity on leaves among plant genotypes (29–31) as well as among species and higher taxonomic ranks (32). Although these patterns are presumably due to phylogenetic variation in ecologically important plant functional traits (33) among host populations and species, the influence of host functional traits on variation in phyllosphere community structure across host species has not been directly quantified. As a result, we have very little understanding of the potential of plant–microbe interaction networks to influence the distribution and functional biogeography of their hosts at large scales in the face of global change (34).A first step toward integrating phyllosphere microbial communities into the study of plant biogeography will require establishing correlations between microbial community structure on leaves and the functional traits of plant hosts. To address this goal, we used high-throughput sequencing to characterize the structure of the bacterial phyllosphere microbiome on the leaves of multiple host tree species in a diverse neotropical forest in Panama. We combined phyllosphere microbiome data with a rich dataset on the attributes of plant hosts, including functional traits and evolutionary relationships, to (i) quantify the magnitude of leaf-associated bacterial biodiversity in a diverse natural forest community, (ii) identify the host plant attributes that influence microbiome community assembly on leaves, and (iii) understand relationships between bacterial biodiversity and suites of host plant traits and functions and discuss their implications for our understanding of plant functional biogeography.     

Dynamic localization of a helper NLR at the plant–pathogen interface underpins pathogen recognition

Plants employ sensor–helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet, the subcellular localization of NLRs pre- and postactivation during pathogen infection remains poorly understood. Here, we show that NRC4, from the “NRC” solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant–pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extrahaustorial membrane (EHM), presumably to mediate response to perihaustorial effectors that are recognized by NRC4-dependent sensor NLRs. However, not all NLRs accumulate at the EHM, as the closely related helper NRC2 and the distantly related ZAR1 did not accumulate at the EHM. NRC4 required an intact N-terminal coiled-coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that postactivation, NRC4 may undergo a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection, however, NRC4 forms puncta mainly at the EHM and, to a lesser extent, at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.

Filamentous pathogens cause devastating diseases on crops, posing a major threat to food security. Some oomycete and fungal pathogens produce specialized hyphal extensions called haustoria that invade the host cells. Haustoria are critical infection structures implicated in delivery of effector proteins and nutrient uptake (1–6). These specialized infection structures are accommodated within the plant cells but are excluded from the host cytoplasm through a newly synthesized membrane called the extrahaustorial membrane (EHM). An intriguing, yet poorly understood, observation is that the EHM is continuous with the host plasma membrane (PM) but is distinct in lipid and protein composition (7, 8). Most of the proteins embedded in the PM, such as surface immune receptors, are excluded from the EHM (9–11). Despite its critical role as the ultimate interface mediating macromolecule exchange between the host and parasite, the mechanisms underlying the biogenesis and functions of the EHM are poorly understood (12).Pathogens deliver effector proteins inside the host cells to neutralize immune responses and enable parasitic infection. A well-studied class of effectors delivered via haustoria are the RXLR family of effectors secreted by Phytophthora infestans (2, 6, 13). RXLR effectors traffic to diverse plant cell compartments to suppress host immunity and mediate nutrient uptake. Remarkably, several P. infestans RXLR effectors focally accumulate at the haustorium interface and perturb cellular defenses (7, 13–15). These include AVRblb2 and AVR1, both of which are implicated in targeting host defense-related secretory pathways to contribute to pathogen virulence (14, 16). Notably, all P. infestans isolates harbor multiple Avrblb2 paralogues (17), which are recognized by the broad-spectrum disease resistance gene Rpi-blb2 cloned from the wild potato species Solanum bulbocastanum (14, 18, 19). On the other hand, AVR1 is sensed by the late blight resistance gene R1, which provides race-specific resistance to AVR1 carrying P. infestans strains (20).Both Rpi-blb2 and R1 encode nucleotide-binding (NB), leucine-rich repeat (LRR) (NLR) proteins, which belong to an NLR network in solanaceae and other asterid plants known as the NLR REQUIRED FOR CELL DEATH (NRC) family. The NRC immune network members form a superclade that consists of about one-third of all Solanaceae NLRs, providing disease resistance to nematodes, viruses, bacteria, oomycetes, and aphids (21). Within the NRC network, sensor NLRs specialized to recognize effectors secreted by pathogens are coupled to helper NLRs (NRCs) that translate the defense signal into disease resistance. We recently showed that Rpi-blb2 and R1 are “sensor NLRs” that require the “helper NLR” NRC4 for the immune-related programmed cell death known as the hypersensitive response (HR) and subsequent disease resistance (21). How and where AVRblb2 and AVR1 are recognized by the NRC4 helper–sensor pairs, as well as the mechanism that leads to HR and disease resistance following their recognition, is unknown. Because AVRblb2 and AVR1 localize to the EHM (13, 14), it is likely that the NLR receptor pairs that sense these perihaustorial effectors also accumulate at the haustorial interface. So far, live-cell imaging of NLRs during infection, which would allow for a greater understanding of NLR functions, has not been feasible due to cell death activation. However, recently solved structures of activated NLRs uncovered critical residues that can be mutated to avoid HR activation without perturbing other NLR functions such as effector recognition and self-oligomerization following activation (22–24). Therefore, fluorescent protein fusions of these NLR mutants could be used for cell biology studies to investigate NLR activities during infection.All NLRs in the NRC superclade carry N-terminal coiled-coil (CC) domains, a characteristic of the CC-NLR type of immune receptors. The recently resolved cryogenic electron microscopy (cryo-EM) structures of activated/nonactivated forms of the CC-NLR type of resistance protein AtZAR1 (22, 25), which provides resistance to several bacterial species, revealed an intriguing model for HR elicitation. Upon activation, AtZAR1 (hereafter, ZAR1) oligomerizes into an inflammasome-like structure, called a “resistosome.” The ZAR1 resistosome consists of five ZAR1 proteins that assemble into a pentameric structure together with the kinases required for ZAR1 activation. Intriguingly, upon immune activation, the first alpha helix (α1) within the CC domain of ZAR1 is exposed and the five α1 helices of the ZAR1 pentamer assemble into a funnel-shaped structure. The resistosome inserts into the PM, forming a pore that could disrupt the cellular integrity or lead to ion flux across the membrane leading to an HR (26, 27). This challenged the long-held view that NLRs execute HR and resistance through activation of downstream signaling cascades. However, whether this model could be applied to other NLRs is still unknown.We recently made an exciting discovery that an N-terminal motif (“the MADA motif”) overlapping ZAR1’s α1 helix, with the consensus sequence MADAxVSFxVxKLxxLLxxEx, exists in ∼20% of CC-NLRs from monocot and dicot species. Remarkably, this motif is preserved in NRC helpers but not in their sensor mates (23). Intriguingly, the first 29 amino acids (aa) of NRC4 containing the MADA motif elicited HR when fused to YFP on its C terminus but not when it is tagged with the YFP mutant that cannot oligomerize. These results indicated that the N terminus of NRC4 relies on a scaffold such as YFP or the rest of NRC4 to form oligomers and trigger cell death. Notably, we previously showed that a chimeric NRC4 construct carrying ZAR1’s α1 helix is functional for triggering HR and confers disease resistance when coexpressed with Rpi-blb2 in lines lacking NRC4 (23), indicating that the proposed ZAR1 mode of action could be applied to NRC helpers.Although recent structural studies greatly improved our understanding of the NLR-mediated immunity and provide unprecedented insights into NLR mode of action, subcellular distribution of NLRs during infection with relevant pathogens is unknown. In the absence of infection, NLRs have been shown to localize to various cellular compartments such as cytoplasm, PM, nucleus, and tonoplast (28–32). These compartments can be required for effector recognition, subsequent HR activation, and/or immune signaling, and some NLRs require a shift in their localization to perceive the effector and initiate the immune responses (30, 32–36). However, determining the subcellular localization of NLRs during infection by relevant microbes has not been feasible due to activation of HR when the corresponding effector is present. Nevertheless, it is possible to monitor the distribution of the NRC helpers in plants that lack the sensor NLRs specialized to recognize the pathogen. The solanaceous model plant Nicotiana benthamiana is an excellent system to study the functioning of the Rpi-blb2-NRC4 pair as it contains a functional NRC4 but lacks specialized sensor NLRs that can recognize P. infestans, whereas transgenic plants carrying Rpi-blb2 are fully resistant to P. infestans (14, 18, 19).Here, we describe the dynamic changes in spatiotemporal localization of NRC4 in response to infection by P. infestans. NRC4 accumulates at the newly synthesized EHM, where the corresponding effectors AVRblb2 and AVR1 are deployed (13, 14). Following immune recognition, the activated receptor reorganizes to form punctate structures that target the cell periphery. Our results indicate that NLRs are not necessarily stationary immune receptors but instead can alter their localizations during infection and may further spread to other cellular membranes from the primary site of activation to boost immune responses.     

Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns

Upon delivery, the neonate is exposed for the first time to a wide array of microbes from a variety of sources, including maternal bacteria. Although prior studies have suggested that delivery mode shapes the microbiota''s establishment and, subsequently, its role in child health, most researchers have focused on specific bacterial taxa or on a single body habitat, the gut. Thus, the initiation stage of human microbiome development remains obscure. The goal of the present study was to obtain a community-wide perspective on the influence of delivery mode and body habitat on the neonate''s first microbiota. We used multiplexed 16S rRNA gene pyrosequencing to characterize bacterial communities from mothers and their newborn babies, four born vaginally and six born via Cesarean section. Mothers’ skin, oral mucosa, and vagina were sampled 1 h before delivery, and neonates’ skin, oral mucosa, and nasopharyngeal aspirate were sampled <5 min, and meconium <24 h, after delivery. We found that in direct contrast to the highly differentiated communities of their mothers, neonates harbored bacterial communities that were undifferentiated across multiple body habitats, regardless of delivery mode. Our results also show that vaginally delivered infants acquired bacterial communities resembling their own mother''s vaginal microbiota, dominated by Lactobacillus, Prevotella, or Sneathia spp., and C-section infants harbored bacterial communities similar to those found on the skin surface, dominated by Staphylococcus, Corynebacterium, and Propionibacterium spp. These findings establish an important baseline for studies tracking the human microbiome''s successional development in different body habitats following different delivery modes, and their associated effects on infant health.     

From the Cover: Gut bacteria facilitate adaptation to crop rotation in the western corn rootworm

Insects are constantly adapting to human-driven landscape changes; however, the roles of their gut microbiota in these processes remain largely unknown. The western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) (Coleoptera: Chrysomelidae) is a major corn pest that has been controlled via annual rotation between corn (Zea mays) and nonhost soybean (Glycine max) in the United States. This practice selected for a “rotation-resistant” variant (RR-WCR) with reduced ovipositional fidelity to cornfields. When in soybean fields, RR-WCRs also exhibit an elevated tolerance of antiherbivory defenses (i.e., cysteine protease inhibitors) expressed in soybean foliage. Here we show that gut bacterial microbiota is an important factor facilitating this corn specialist’s (WCR''s) physiological adaptation to brief soybean herbivory. Comparisons of gut microbiota between RR- and wild-type WCR (WT-WCR) revealed concomitant shifts in bacterial community structure with host adaptation to soybean diets. Antibiotic suppression of gut bacteria significantly reduced RR-WCR tolerance of soybean herbivory to the level of WT-WCR, whereas WT-WCR were unaffected. Our findings demonstrate that gut bacteria help to facilitate rapid adaptation of insects in managed ecosystems.     

Encapsulation and characterization of proton-bound amine homodimers in a water-soluble,self-assembled supramolecular host

Cyclic amines can be encapsulated in a water-soluble self-assembled supramolecular host upon protonation. The hydrogen-bonding ability of the cyclic amines, as well as the reduced degrees of rotational freedom, allows for the formation of proton-bound homodimers inside of the assembly that are otherwise not observable in aqueous solution. The generality of homodimer formation was explored with small N-alkyl aziridines, azetidines, pyrrolidines, and piperidines. Proton-bound homodimer formation is observed for N-alkylaziridines (R = methyl, isopropyl, tert-butyl), N-alkylazetidines (R = isopropyl, tert-butyl), and N-methylpyrrolidine. At high concentration, formation of a proton-bound homotrimer is observed in the case of N-methylaziridine. The homodimers stay intact inside the assembly over a large concentration range, thereby suggesting cooperative encapsulation. Both G3(MP2)B3 and G3B3 calculations of the proton-bound homodimers were used to investigate the enthalpy of the hydrogen bond in the proton-bound homodimers and suggest that the enthalpic gain upon formation of the proton-bound homodimers may drive guest encapsulation.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Shigella impairs T lymphocyte dynamics in vivo

The Gram-negative enteroinvasive bacterium Shigella flexneri is responsible for the endemic form of bacillary dysentery, an acute rectocolitis in humans. S. flexneri uses a type III secretion system to inject effector proteins into host cells, thus diverting cellular functions to its own benefit. Protective immunity to reinfection requires several rounds of infection to be elicited and is short-lasting, suggesting that S. flexneri interferes with the priming of specific immunity. Considering the key role played by T-lymphocyte trafficking in priming of adaptive immunity, we investigated the impact of S. flexneri on T-cell dynamics in vivo. By using two-photon microscopy to visualize bacterium–T-cell cross-talks in the lymph nodes, where the adaptive immunity is initiated, we provide evidence that S. flexneri, via its type III secretion system, impairs the migration pattern of CD4⁺ T cells independently of cognate recognition of bacterial antigens. We show that bacterial invasion of CD4⁺ T lymphocytes occurs in vivo, and results in cell migration arrest. In the absence of invasion, CD4⁺ T-cell migration parameters are also dramatically altered. Signals resulting from S. flexneri interactions with subcapsular sinus macrophages and dendritic cells, and recruitment of polymorphonuclear cells are likely to contribute to this phenomenon. These findings indicate that S. flexneri targets T lymphocytes in vivo and highlight the role of type III effector secretion in modulating host adaptive immune responses.     

Porcine Intestinal Organoids: Overview of the State of the Art

The intestinal tract is a crucial part of the body for growth and development, and its dysregulation can cause several diseases. The lack of appropriate in vitro models hampers the development of effective preventions and treatments against these intestinal tract diseases. Intestinal organoids are three-dimensional (3D) polarized structures composed of different types of cells capable of self-organization and self-renewal, resembling their organ of origin in architecture and function. Porcine intestinal organoids (PIOs) have been cultured and are used widely in agricultural, veterinary, and biomedical research. Based on the similarity of the genomic sequence, anatomic morphology, and drug metabolism with humans and the difficulty in obtaining healthy human tissue, PIOs are also considered ideal models relative to rodents. In this review, we summarize the current knowledge on PIOs, emphasizing their culturing, establishment and development, and applications in the study of host–microbe interactions, nutritional development, drug discovery, and gene editing potential.     

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo–cross-linker p-azido-l-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.Introducing light-sensitive moieties into proteins provides a powerful approach to investigate molecular mechanisms as well as biological functions with high temporal and spatial resolution (1, 2). An attractive strategy to engineer light responsiveness relies on the use of photoreactive unnatural amino acids (UAAs), allowing site-specific incorporation in a protein target. The methodology relies on the read-through of an unassigned codon (commonly the amber stop codon) in an mRNA by a suppressor tRNA aminoacylated with a desired UAA. Using this approach, UAAs with unique chemical functionalities including light-sensitivity have been successfully incorporated into ion channels and neurotransmitter receptors, significantly contributing to our understanding of receptor function (3, 4). However, the challenging synthesis of the chemically acylated tRNA has limited the general applicability of the approach. The recent development of genetically engineered suppressor tRNA/aminoacyl-tRNA synthetase pairs with altered amino acid specificity allowed for aminoacylation in the expression system in situ. This method provided a major step forward by advancing the UAA technology to the all-genetic–based level, also known as “the genetic-code expansion” (5–7).Here, we present the design of a light-sensitive ionotropic glutamate receptor (iGluR) through the genetic incorporation of a photoreactive UAA. Our approach takes advantage of the recent development of the genetic-code expansion in Xenopus oocytes (8), which is a classical vehicle for heterologous expression and functional characterization of ligand-gated ion channels (LGICs). We focused on NMDA receptors (NMDARs), which play pivotal roles in brain physiology and pathology (9). NMDARs are obligatory heterotetramers commonly composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Although GluN1 is encoded by a single gene, there are four types of GluN2 subunits (GluN2A to -D) encoded by four different genes, which endow NMDARs with different properties including channel open probabilities (P_o) and sensitivities to allosteric modulators (9). The extracellular region of both GluN1 and GluN2 subunits consists of a tandem of large clamshell-like domains comprising an N-terminal domain (NTD) and an agonist-binding domain (ABD) (Fig. 1A). Besides having essential functions in receptor assembly (10, 11), recent studies of the NTDs have also revealed that the individual GluN2 (12–14) and GluN1 (15) NTDs fine-tune NMDAR gating and pharmacological properties by undergoing large-range conformational changes. The recent X-ray crystal structure of a GluN1/GluN2B NTD complex reveals a unique arrangement of the two NTD protomers with intersubunit interactions distinct from those observed in AMPA and kainate receptors (16, 17). However, the importance of these dimer interfaces in the subunit-specific receptor regulation is poorly understood. We show that encoding the photoreactive UAA p-azido-l-phenylalanine (AzF) at the NTD upper lobe dimer interface in GluN1/GluN2B receptors serves as a photoswitch, triggering irreversible decrease of channel activity upon UV exposure. We further investigated the photo-induced conformational changes at the NTD dimer interfaces, as well as the subunit-dependent regulation, identifying structural determinants that differ between GluN2A- and GluN2B-containing NMDARs. Finally, we applied our approach to mammalian cells, including cultured hippocampal neurons, providing evidence for the transferability of light-sensitive NMDARs to more native cellular environments. Our results not only prove the feasibility of designing light-controlled NMDARs by introducing a genetically encoded photoreactive UAA at a conformational sensitive site, but also reveal aspects of the NMDAR assembly as highly subtype specific.Open in a separate windowFig. 1.Light inactivation of GluN1/GluN2B NMDARs incorporating a genetically encoded photoactive UAA. (A, Left) Four plasmids encoding the GluN1 subunit with an amber stop codon at position Y109 (red dot), the wt GluN2 subunit, the suppressor tRNA (Yam), and the engineered tRNA synthetase (RS) were coinjected into Xenopus oocytes. (Center) Crystal structure of the GluA2 AMPA receptor (40). The three major domains—N-terminal domain (NTD), agonist-binding domain (ABD) and transmembrane domain (TMD)—are arranged in layers. One NTD dimer is highlighted. (Right) Crystal structure of the NMDAR GluN1/GluN2B NTD heterodimer (16); the ifenprodil molecule is omitted for clarity. LL, lower lobe; UL, upper lobe. The GluN1-Y109 site is highlighted. On UV irradiation, the azide moiety generates a biradical, which subsequently can react with a nearby residue to form a covalent adduct. (B) Current amplitudes from oocytes injected with plasmids as indicated, in the absence or presence of UAAs. For each condition, 20 oocytes were tested. Only currents >10 nA were plotted. (C) Representative current traces showing UV-induced current inhibition of GluN1-Y109AzF/GluN2B receptors but not wt GluN1/GluN2B receptors. (D) UV-induced current modifications at wt GluN1/GluN2B (1.11 ± 0.13; n = 8), GluN1-Y109AzF/GluN2Bwt with (0.28 ± 0.05; n = 16) or without (0.31 ± 0.05; n = 5) agonist, and GluN1-Y109Bpa/GluN2Bwt (1.15 ± 0.06; n = 5) receptors. Error bars, SD. (E) MK-801 inhibition kinetics of wt GluN1/GluN2B and GluN1-Y109AzF/GluN2B receptors before and after UV treatment.     

The bacterial signal indole increases epithelial-cell tight-junction resistance and attenuates indicators of inflammation

Interkingdom signaling is established in the gastrointestinal tract in that human hormones trigger responses in bacteria; here, we show that the corollary is true, that a specific bacterial signal, indole, is recognized as a beneficial signal in intestinal epithelial cells. Our prior work has shown that indole, secreted by commensal Escherichia coli and detected in human feces, reduces pathogenic E. coli chemotaxis, motility, and attachment to epithelial cells. However, the effect of indole on intestinal epithelial cells is not known. Because intestinal epithelial cells are likely to be exposed continuously to indole, we hypothesized that indole may be beneficial for these cells, and investigated changes in gene expression with the human enterocyte cell line HCT-8 upon exposure to indole. Exposure to physiologically relevant amounts of indole increased expression of genes involved in strengthening the mucosal barrier and mucin production, which were consistent with an increase in the transepithelial resistance of HCT-8 cells. Indole also decreased TNF-α-mediated activation of NF-κB, expression of the proinflammatory chemokine IL-8, and the attachment of pathogenic E. coli to HCT-8 cells, as well as increased expression of the antiinflammatory cytokine IL-10. The changes in transepithelial resistance and NF-κB activation were specific to indole: other indole-like molecules did not elicit a similar response. Our results are similar to those observed with probiotic strains and suggest that indole could be important in the intestinal epithelial cells response to gastrointestinal tract pathogens.     

Multipoint molecular recognition within a calix[6]arene funnel complex

A multipoint recognition system based on a calix[6]arene is described. The calixarene core is decorated on alternating aromatic subunits by 3 imidazole arms at the small rim and 3 aniline groups at the large rim. This substitution pattern projects the aniline nitrogens toward each other when Zn(II) binds at the Tris-imidazole site or when a proton binds at an aniline. The XRD structure of the monoprotonated complex having an acetonitrile molecule bound to Zn(II) in the cavity revealed a constrained geometry at the metal center reminiscent of an entatic state. Computer modeling suggests that the aniline groups behave as a tritopic monobasic site in which only 1 aniline unit is protonated and interacts with the other 2 through strong hydrogen bonding. The metal complex selectively binds a monoprotonated diamine vs. a monoamine through multipoint recognition: coordination to the metal ion at the small rim, hydrogen bonding to the calix-oxygen core, CH/π interaction within the cavity''s aromatic walls, and H-bonding to the anilines at the large rim.     

Deconstructing the Phage–Bacterial Biofilm Interaction as a Basis to Establish New Antibiofilm Strategies

The bacterial biofilm constitutes a complex environment that endows the bacterial community within with an ability to cope with biotic and abiotic stresses. Considering the interaction with bacterial viruses, these biofilms contain intrinsic defense mechanisms that protect against phage predation; these mechanisms are driven by physical, structural, and metabolic properties or governed by environment-induced mutations and bacterial diversity. In this regard, horizontal gene transfer can also be a driver of biofilm diversity and some (pro)phages can function as temporary allies in biofilm development. Conversely, as bacterial predators, phages have developed counter mechanisms to overcome the biofilm barrier. We highlight how these natural systems have previously inspired new antibiofilm design strategies, e.g., by utilizing exopolysaccharide degrading enzymes and peptidoglycan hydrolases. Next, we propose new potential approaches including phage-encoded DNases to target extracellular DNA, as well as phage-mediated inhibitors of cellular communication; these examples illustrate the relevance and importance of research aiming to elucidate novel antibiofilm mechanisms contained within the vast set of unknown ORFs from phages.