Intercellular communication is required for trap formation in the nematode-trapping fungus Duddingtonia flagrans

Nematode-trapping fungi (NTF) are a large and diverse group of fungi, which may switch from a saprotrophic to a predatory lifestyle if nematodes are present. Different fungi have developed different trapping devices, ranging from adhesive cells to constricting rings. After trapping, fungal hyphae penetrate the worm, secrete lytic enzymes and form a hyphal network inside the body. We sequenced the genome of Duddingtonia flagrans, a biotechnologically important NTF used to control nematode populations in fields. The 36.64 Mb genome encodes 9,927 putative proteins, among which are more than 638 predicted secreted proteins. Most secreted proteins are lytic enzymes, but more than 200 were classified as small secreted proteins (< 300 amino acids). 117 putative effector proteins were predicted, suggesting interkingdom communication during the colonization. As a first step to analyze the function of such proteins or other phenomena at the molecular level, we developed a transformation system, established the fluorescent proteins GFP and mCherry, adapted an assay to monitor protein secretion, and established gene-deletion protocols using homologous recombination or CRISPR/Cas9. One putative virulence effector protein, PefB, was transcriptionally induced during the interaction. We show that the mature protein is able to be imported into nuclei in Caenorhabditis elegans cells. In addition, we studied trap formation and show that cell-to-cell communication is required for ring closure. The availability of the genome sequence and the establishment of many molecular tools will open new avenues to studying this biotechnologically relevant nematode-trapping fungus

Predatory fungi with pest control potential:

Nematoden-fangende Pilze kommen ubiquitär vor allem in Böden vor. Sie leben von abgestorbenem, organischem Material, können aber eine räuberische Lebensweise einschlagen, wenn die Nährstoffe knapp werden. Dann bilden sie je nach Spezies unterschiedliche Fallentypen aus. Arthrobotrys flagrans bildet klebrige Fallennetzwerke. Die Fallenbildung wird durch das Zusammenspiel mehrerer Nematoden-eigener Pheromone und pilzlichen Signalstoffen reguliert. Wenn sich ein Nematode in dem Fallennetzwerk verfangen hat, dringt eine Penetrationshyphe durch die Cuticula und die Epidermis in den Wurmkörper ein. Dort verdickt sich die Hyphe zu einem Bulbus und wächst als Ernährungshyphe durch den Nematodenkörper. Durch lytische Enzyme wird der tierische Körper zersetzt und die Nährstoffe vom Pilz aufgenommen. Im späten Stadium der Attacke wachsen Hyphen aus dem Nematoden in die Umgebung aus. Bei der Penetration und vermutlich auch bei der weiteren Besiedlung des Nematoden spielen kleine, sekretierte pilzliche Proteine eine wichtige Rolle als Virulenzfaktoren. Die Nematoden-Pilz-Interaktion ist nicht nur ein faszinierendes Grundlagenforschungsgebiet, sondern die Pilze können auch zur Bekämpfung schädlicher Nematoden eingesetzt werden.Nematode-trapping fungi are ubiquitous, especially in soil. They live on dead, organic matter, but can adopt a predatory lifestyle when nutrients become scarce. Then they form different types of traps depending on the species. Arthrobotrys flagrans forms sticky trap networks. The formation of traps is regulated by the interaction of several nematode-specific pheromones and fungal signaling molecules. When a nematode becomes entangled in the trap network, a penetrating hypha enters through the cuticle and epidermis into the nematode body. There the hypha thickens into a bulb and grows through the nematode body as a trophic hypha. The animal body is decomposed by lytic enzymes and the nutrients are absorbed by the fungus. In the late stage of the attack, hyphae grow out of the nematode into the environment. Small, secreted fungal proteins play an important role as virulence factors during penetration and presumably also during further colonization of the nematode. Not only is the nematode fungus interaction a fascinating area of basic research, but the fungi can also be used to control harmful nematodes

Synchronization of oscillatory growth prepares fungal hyphae for fusion

Communication is crucial for organismic interactions, from bacteria, to fungi, to humans. Humans may use the visual sense to monitor the environment before starting acoustic interactions. In comparison, fungi, lacking a visual system, rely on a cell-to-cell dialogue based on secreted signaling molecules to coordinate cell fusion and establish hyphal networks. Within this dialogue, hyphae alternate between sending and receiving signals. This pattern can be visualized via the putative signaling protein Soft (SofT), and the mitogen-activated protein kinase MAK-2 (MakB) which are recruited in an alternating oscillatory manner to the respective cytoplasmic membrane or nuclei of interacting hyphae. Here, we show that signal oscillations already occur in single hyphae of Arthrobotrys flagrans in the absence of potential fusion partners (cell monologue). They were in the same phase as growth oscillations. In contrast to the anti-phasic oscillations observed during the cell dialogue, SofT and MakB displayed synchronized oscillations in phase during the monologue. Once two fusion partners came into each other’s vicinity, their oscillation frequencies slowed down (entrainment phase) and transit into anti-phasic synchronization of the two cells’ oscillations with frequencies of 104±28 s and 117±19 s, respectively. Single-cell oscillations, transient entrainment, and anti-phasic oscillations were reproduced by a mathematical model where nearby hyphae can absorb and secrete a limited molecular signaling component into a shared extracellular space. We show that intracellular Ca2+ concentrations oscillate in two approaching hyphae, and depletion of Ca2+ from the medium affected vesicle-driven extension of the hyphal tip, abolished the cell monologue and the anti-phasic synchronization of two hyphae. Our results suggest that single hyphae engage in a ‘monologue’ that may be used for exploration of the environment and can dynamically shift their extracellular signaling systems into a ‘dialogue’ to initiate hyphal fusion

Structural responses of model biomembranes to Mars-relevant salts

Lipid membranes are a key component of contemporary living systems and are thought to have been essential to the origin of life. Most research on membranes has focused on situations restricted to ambient physiological or benchtop conditions. However, the influence of more extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments are less well understood. The deep subsurface environments of Mars, for instance, may harbor high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments for cellular life. Here, we investigated the combined effects of high concentrations of salts, including sodium and magnesium perchlorate and sulfate, and high hydrostatic pressure on the stability and structure of model biomembranes of varying complexity. To this end, a variety of biophysical techniques have been applied, which include calorimetry, fluorescence spectroscopies, small-angle X-ray scattering, dynamic light scattering, and microscopy techniques. We show that the structure and phase behavior of lipid membranes is sensitively dictated by the nature of the salt, in particular its anion and its concentration. We demonstrate that, with the exception of magnesium perchlorate, which can also induce cubic lipid arrangements, long-chain saturated lipid bilayer structures can still persist at high salt concentrations across a range of pressures. The lateral organization of complex heterogeneous raft-like membranes is affected by all salts. For simple, in particular bacterial membrane-type bilayer systems with unsaturated chains, vesicular structures are still stable at Martian brine conditions, also up to the kbar pressure range, demonstrating the potential compatibility of environments containing such ionic and pressure extremes to lipid-encapsulated life