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Allelochemicals represent a class of natural products released by plants as root, leaf, and fruit exudates that interfere with the growth and survival of neighboring plants. Understanding how allelochemicals function to regulate plant responses may provide valuable new approaches to better control plant function. One such allelochemical, Myrigalone A (MyA) produced by Myrica gale, inhibits seed germination and seedling growth through an unknown mechanism. Here, we investigate MyA using the tractable model Dictyostelium discoideum and reveal that its activity depends on the conserved homolog of the plant ethylene synthesis protein 1-aminocyclopropane-1-carboxylic acid oxidase (ACO). Furthermore, in silico modeling predicts the direct binding of MyA to ACO within the catalytic pocket. In D. discoideum, ablation of ACO mimics the MyA-dependent developmental delay, which is partially restored by exogenous ethylene, and MyA reduces ethylene production. In Arabidopsis thaliana, MyA treatment delays seed germination, and this effect is rescued by exogenous ethylene. It also mimics the effect of established ACO inhibitors on root and hypocotyl extension, blocks ethylene-dependent root hair production, and reduces ethylene production. Finally, in silico binding analyses identify a range of highly potent ethylene inhibitors that block ethylene-dependent response and reduce ethylene production in Arabidopsis. Thus, we demonstrate a molecular mechanism by which the allelochemical MyA reduces ethylene biosynthesis and identify a range of ultrapotent inhibitors of ethylene-regulated responses.

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Organisms often cooperate through the production of freely available public goods. This can greatly benefit the group but is vulnerable to the "tragedy of the commons" if individuals lack the motivation to make the necessary investment into public goods production. Relatedness to groupmates can motivate individual investment because group success ultimately benefits their genes' own self-interests. However, systems often lack mechanisms that can reliably ensure that relatedness is high enough to promote cooperation. Consequently, groups face a persistent threat from the tragedy unless they have a mechanism to enforce investment when relatedness fails to provide adequate motivation. To understand the real threat posed by the tragedy and whether groups can avert its impact, we determine how the social amoeba Dictyostelium discoideum responds as relatedness decreases to levels that should induce the tragedy. We find that, while investment in public goods declines as overall within-group relatedness declines, groups avert the expected catastrophic collapse of the commons by continuing to invest, even when relatedness should be too low to incentivize any contribution. We show that this is due to a developmental buffering system that generates enforcement because insufficient cooperation perturbs the balance of a negative feedback system controlling multicellular development. This developmental constraint enforces investment under the conditions expected to be most tragic, allowing groups to avert a collapse in cooperation. These results help explain how mechanisms that suppress selfishness and enforce cooperation can arise inadvertently as a by-product of constraints imposed by selection on different traits.

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Natural selection should favour generalist predators that outperform specialists across all prey types. Two genetic solutions could explain why intraspecific variation in predatory performance is, nonetheless, widespread: mutations beneficial on one prey type are costly on another (antagonistic pleiotropy), or mutational effects are prey-specific, which weakens selection, allowing variation to persist (relaxed selection). To understand the relative importance of these alternatives, we characterised natural variation in predatory performance in the microbial predator Dictyostelium discoideum. We found widespread nontransitive differences among strains in predatory success across different bacterial prey, which can facilitate stain coexistence in multi-prey environments. To understand the genetic basis, we developed methods for high throughput experimental evolution on different prey (REMI-seq). Most mutations (~77%) had prey-specific effects, with very few (~4%) showing antagonistic pleiotropy. This highlights the potential for prey-specific effects to dilute selection, which would inhibit the purging of variation and prevent the emergence of an optimal generalist predator.

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BACKGROUND: Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. RESULTS: We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of 'barcoded' mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. CONCLUSIONS: We present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.

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A critical aspect of toxicity evaluation is developmental and reproductive toxicity (DART) testing. Traditionally, DART testing has been conducted in vivo in mammalian model systems. New legislation aimed at reducing animal use and the prohibitive costs associated with DART testing, together with a need to understand the genetic pathways underlying developmental toxicity means there is a growing demand for alternative model systems for toxicity evaluation. Here we explore the potential of the eukaryotic social amoeba Dictyostelium discoideum, which is already widely used as a simple model system for cell and developmental biology, as a potential nonanimal model for DART testing. We developed assays for high-throughput screening of toxicity during D. discoideum growth and development. This allowed the toxicity of a broad range of test compounds to be characterized, which revealed that D. discoideum can broadly predict mammalian toxicity. In addition, we show that this system can be used to perform functional genomic screens to compare the molecular modes of action of different compounds. For example, genome-wide screens for mutations that affect lithium and valproic acid toxicity allowed common and unique biological targets and molecular processes mediating their toxicity to be identified. These studies illustrate that D. discoideum could represent a predictive nonanimal model for DART testing due to its amenability to high-throughput approaches and molecular genetic tractability.

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Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify "adaptive codon preference," a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated "preference" largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome.

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Conflict is thought to play a critical role in the evolution of social interactions by promoting diversity or driving accelerated evolution. However, despite our sophisticated understanding of how conflict shapes social traits, we have limited knowledge of how it impacts molecular evolution across the underlying social genes. Here we address this problem by analyzing the genome-wide impact of social interactions using genome sequences from 67 Dictyostelium discoideum strains. We find that social genes tend to exhibit enhanced polymorphism and accelerated evolution. However, these patterns are not consistent with conflict driven processes, but instead reflect relaxed purifying selection. This pattern is most likely explained by the conditional nature of social interactions, whereby selection on genes expressed only in social interactions is diluted by generations of inactivity. This dilution of selection by inactivity enhances the role of drift, leading to increased polymorphism and accelerated evolution, which we call the Red King process.

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Dictyostelium discoideum (D. discoideum) is a simple eukaryote with a unique life cycle in which it differentiates from unicellular amoebae into a fruiting body upon starvation. Reactive oxygen species (ROS) have been associated with bacterial predation, as well as regulatory events during D. discoideum development and differentiation. Coenzyme A (CoA) is a key metabolic integrator in all living cells. A novel function of CoA in redox regulation, mediated by covalent attachment of CoA to cellular proteins in response to oxidative or metabolic stress, has been recently discovered and termed protein CoAlation. In this study, we report that the level of CoA and protein CoAlation in D. discoideum are developmentally regulated, and correlate with the temporal expression pattern of genes implicated in CoA biosynthesis during morphogenesis. Furthermore, treatment of growing D. discoideum cells with oxidising agents results in a dose-dependent increase of protein CoAlation. However, much higher concentrations were required when compared to mammalian cells and bacteria. Increased resistance of D. discoideum to oxidative stress induced by H2O2 has previously been attributed to high levels of catalase activity. In support of this notion, we found that H2O2-induced protein CoAlation is significantly increased in CatA-deficient D. discoideum cells. Collectively, this study provides insights into the role of CoA and protein CoAlation in the maintenance of redox homeostasis in amoeba and during D. discoideum morphogenesis.

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Cell-cell heterogeneity can facilitate lineage choice during embryonic development because it primes cells to respond to differentiation cues. However, remarkably little is known about the origin of heterogeneity or whether intrinsic and extrinsic variation can be controlled to generate reproducible cell type proportioning seen in vivo. Here, we use experimentation and modeling in D. discoideum to demonstrate that population-level cell cycle heterogeneity can be optimized to generate robust cell fate proportioning. First, cell cycle position is quantitatively linked to responsiveness to differentiation-inducing signals. Second, intrinsic variation in cell cycle length ensures cells are randomly distributed throughout the cell cycle at the onset of multicellular development. Finally, extrinsic perturbation of optimal cell cycle heterogeneity is buffered by compensatory changes in global signal responsiveness. These studies thus illustrate key regulatory principles underlying cell-cell heterogeneity optimization and the generation of robust and reproducible fate choice in development.

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Contributing to cooperation is typically costly, while its rewards are often available to all members of a social group. So why should individuals be willing to pay these costs, especially if they could cheat by exploiting the investments of others? Kin selection theory broadly predicts that individuals should invest more into cooperation if their relatedness to group members is high (assuming they can discriminate kin from nonkin). To better understand how relatedness affects cooperation, we derived the ?Collective Investment" game, which provides quantitative predictions for patterns of strategic investment depending on the level of relatedness. We then tested these predictions by experimentally manipulating relatedness (genotype frequencies) in mixed cooperative aggregations of the social amoeba Dictyostelium discoideum, which builds a stalk to facilitate spore dispersal. Measurements of stalk investment by natural strains correspond to the predicted patterns of relatedness-dependent strategic investment, wherein investment by a strain increases with its relatedness to the group. Furthermore, if overall group relatedness is relatively low (i.e., no strain is at high frequency in a group) strains face a scenario akin to the "Prisoner's Dilemma" and suffer from insufficient collective investment. We find that strains employ relatedness-dependent segregation to avoid these pernicious conditions. These findings demonstrate that simple organisms like D. discoideum are not restricted to being ?cheaters" or ?cooperators" but instead measure their relatedness to their group and strategically modulate their investment into cooperation accordingly. Consequently, all individuals will sometimes appear to cooperate and sometimes cheat due to the dynamics of strategic investing.

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Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using in silico docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds.

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Cheaters disrupt cooperation by reaping the benefits without paying their fair share of associated costs. Cheater impact can be diminished if cooperators display a tag ('greenbeard') and recognise and preferentially direct cooperation towards other tag carriers. Despite its popular appeal, the feasibility of such greenbeards has been questioned because the complex patterns of partner-specific cooperative behaviours seen in nature require greenbeards to come in different colours. Here we show that a locus ('Tgr') of a social amoeba represents a polychromatic greenbeard. Patterns of natural Tgr locus sequence polymorphisms predict partner-specific patterns of cooperation by underlying variation in partner-specific protein-protein binding strength and recognition specificity. Finally, Tgr locus polymorphisms increase fitness because they help avoid potential costs of cooperating with incompatible partners. These results suggest that a polychromatic greenbeard can provide a key mechanism for the evolutionary maintenance of cooperation.

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Interaction conditions can change the balance of cooperation and conflict in multicellular groups. After aggregating together, cells of the social amoeba Dictyostelium discoideum may migrate as a group (known as a slug) to a new location. We consider this migration stage as an arena for social competition and conflict because the cells in the slug may not be from a genetically homogeneous population. In this study, we examined the interplay of two seemingly diametric actions, the solitary action of kin recognition and the collective action of slug migration in D. discoideum, to more fully understand the effects of social competition on fitness over the entire lifecycle. We compare slugs composed of either genetically homogenous or heterogeneous cells that have migrated or remained stationary in the social stage of the social amoeba Dictyostelium discoideum. After migration of chimeric slugs, we found that facultative cheating is reduced, where facultative cheating is defined as greater contribution to spore relative to stalk than found for that clone in the clonal state. In addition our results support previous findings that competitive interactions in chimeras diminish slug migration distance. Furthermore, fruiting bodies have shorter stalks after migration, even accounting for cell numbers at that time. Taken together, these results show that migration can alleviate the conflict of interests in heterogeneous slugs. It aligns their interest in finding a more advantageous place for dispersal, where shorter stalks suffice, which leads to a decrease in cheating behavior.

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Cooperation is ubiquitous across the tree of life, from simple microbes to the complex social systems of animals. Individuals cooperate by engaging in costly behaviors that can be exploited by other individuals who benefit by avoiding these associated costs. Thus, if successful exploitation of social partners during cooperative interactions increases relative fitness, then we expect selection to lead to the emergence of a single optimal winning strategy in which individuals maximize their gain from cooperation while minimizing their associated costs. Such social "cheating" appears to be widespread in nature, including in several microbial systems, but despite the fitness advantages favoring social cheating, populations tend to harbor significant variation in social success rather than a single optimal winning strategy. Using the social amoeba Dictyostelium discoideum, we provide a possible explanation for the coexistence of such variation. We find that genotypes typically designated as "cheaters" because they produce a disproportionate number of spores in chimeric fruiting bodies do not actually gain higher fitness as a result of this apparent advantage because they produce smaller, less viable spores than putative "losers." As a consequence of this trade-off between spore number and viability, genotypes with different spore production strategies, which give the appearance of differential social success, ultimately have similar realized fitness. These findings highlight the limitations of using single fitness proxies in evolutionary studies and suggest that interpreting social trait variation in terms of strategies like cheating or cooperating may be misleading unless these behaviors are considered in the context of the true multidimensional nature of fitness.

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BACKGROUND: Dictyostelium discoideum, a microbial model for social evolution, is known to distinguish self from non-self and show genotype-dependent behavior during chimeric development. Aside from a small number of cell-cell recognition genes, however, little is known about the genetic basis of self/non-self recognition in this species. Based on the key hypothesis that there should be differential expression of genes if D. discoideum cells were interacting with non-clone mates, we performed transcriptomic profiling study in this species during clonal vs. chimeric development. The transcriptomic profiles of D. discoideum cells in clones vs. different chimeras were compared at five different developmental stages using a customized microarray. Effects of chimerism on global transcriptional patterns associated with social interactions were observed. RESULTS: We find 1,759 genes significantly different between chimera and clone, 1,144 genes associated significant strain differences, and 6,586 genes developmentally regulated over time. Principal component analysis showed a small amount of the transcriptional variance to chimerism-related factors (Chimerism: 0.18%, Chimerism × Timepoint: 0.03%). There are 162 genes specifically regulated under chimeric development, with continuous small differences between chimera vs. clone over development. Almost 60% of chimera-associated differential genes were differentially expressed at the 4 h aggregate stage, which corresponds to the initial transition of D. discoideum from solitary life to a multicellular phase. CONCLUSIONS: A relatively small proportion of over-all variation in gene expression is explained by differences between chimeric and clonal development. The relatively small modifications in gene expression associated with chimerism is compatible with the high level of cooperation observed among different strains of D. discoideum; cells of distinct genetic backgrounds will co-aggregate indiscriminately and co-develop into fruiting bodies. Chimeric development may involve re-programming of the transcriptome through small modifications of the developmental genetic network, which may also indicate that response to social interaction involves many genes with individually small transcriptional effect.

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Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF-hand-containing Rab GAP found in a complex with Rab11a and P2XA. CnrF Rab GAP activity for Rab11a is enhanced by the presence of calcium and the EF-hand domain. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion.

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In cell culture, genetically identical cells often exhibit heterogeneous behavior, with only 'lineage primed' cells responding to differentiation inducing signals. It has recently been proposed that such heterogeneity exists during normal embryonic development to allow position independent patterning based on 'salt and pepper' differentiation and sorting out. However, the molecular basis of lineage priming and how it leads to reproducible cell type proportioning are poorly understood. To address this, we employed a novel forward genetic approach in the model organism Dictyostelium discoideum. These studies reveal that the Ras-GTPase regulator gefE is required for normal lineage priming and salt and pepper differentiation. This is because Ras-GTPase activity sets the intrinsic response threshold to lineage specific differentiation signals. Importantly, we show that although gefE expression is uniform, transcription of its target, rasD, is both heterogeneous and dynamic, thus providing a novel mechanism for heterogeneity generation and position-independent differentiation. DOI: http://dx.doi.org/10.7554/eLife.01067.001.

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The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.

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One condition for the evolution of altruism is genetic relatedness between altruist and beneficiary, often achieved through active kin recognition. Here, we investigate the power of a passive process resulting from genetic drift during population growth in the social amoeba Dictyostelium discoideum. We put labelled and unlabelled cells of the same clone in the centre of a plate, and allowed them to proliferate outward. Zones formed by genetic drift owing to the small population of actively growing cells at the colony edge. We also found that single cells could form zones of high relatedness. Relatedness increased at a significantly higher rate when food was in short supply. This study shows that relatedness can be significantly elevated before the social stage without a small founding population size or recognition mechanism.

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From microbes to metazoans, it is now clear that fluctuations in the abundance of mRNA transcripts and protein molecules enable genetically identical cells to oscillate between several distinct states (Kaern et al. 2005). Since this cell-cell variability does not derive from physical differences in the genetic code it is termed non-genetic heterogeneity. Non-genetic heterogeneity endows cell populations with useful capabilities they could never achieve if each cell were the same as its neighbors (Raj & van Oudenaarden 2008; Eldar & Elowitz 2010). One such example is seen during multicellular development and "salt and pepper" cell type differentiation. In this review, we will first examine the importance of non-genetic heterogeneity in initiating "salt and pepper" pattern formation during Dictyostelium discoideum development. Second, we will discuss the various ways in which non-genetic heterogeneity might be generated, as well as recent advances in understanding the molecular basis of heterogeneity in this system.

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