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The fungus Fusarium oxysporum (Fo) is widely known for causing wilt disease in over 100 different plant species. Endophytic interactions of Fo with plants are much more common, and strains pathogenic on one plant species can even be beneficial endophytes on another species. However, endophytic and beneficial interactions have been much less investigated at the molecular level, and the genetic basis that underlies endophytic versus pathogenic behavior is unknown. To investigate this, 44 Fo strains from non-cultivated Australian soils, grass roots from Spain, and tomato stems from United States were characterized genotypically by whole genome sequencing, and phenotypically by examining their ability to symptomlessly colonize tomato plants and to confer resistance against Fusarium Wilt. Comparison of the genomes of the validated endophytic Fo strains with those of 102 pathogenic strains revealed that both groups have similar genomes sizes, with similar amount of accessory DNA. However, although endophytic strains can harbor homologs of known effector genes, they have typically fewer effector gene candidates and associated non-autonomous transposons (mimps) than pathogenic strains. A pathogenic ‘lifestyle’ is associated with extended effector gene catalogs and a set of “host specific” effectors. No candidate effector genes unique to endophytic strains isolated from the same plant species were found, implying little or no host-specific adaptation. As plant-beneficial interactions were observed to be common for the tested Fo isolates, the propensity for endophytism and the ability to confer biocontrol appears to be a predominant feature of this organism. These findings allow prediction of the lifestyle of a Fo strain based on its genome sequence as a potential pathogen or as a harmless or even beneficial endophyte by determining its effectorome and mimp number.

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go–related gene) channels and calcium (Ca2+)–activated big-conductance potassium (BK)–type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.

Plasma membrane Ca2+-ATPases (PMCA), or Ca2+-pumps, terminate Ca2+-signals in any type of cell by extruding Ca2+ from the cytosol to the extracellular space. In neurons and epithelial cells, Ca2+-extrusion occurs within tens of milliseconds, a speed that is not compatible with current knowledge on PMCA activity. Here we investigated the transport velocity of Ca2+ -pumps assembled from PMCA2 and the recently identified auxiliary subunit Neuroplastin in intact cells. Using Ca2+-activated K+ channels as fast reporters, we show that PMCA2-Neuroplastin complexes, the most abundant Ca2+-transporters in the mammalian brain, provide Ca2+-clearing in the low millisecond-range. Freeze-fracture derived immuno-EM data on densities of Ca2+-source(s) and Ca2+-transporters translated these kinetics into transport rates for PMCA2-Neuroplastin complexes of more than 6000 cycles/s. Direct comparison with the Na+/Ca2+-exchanger NCX2, an alternate-access transporter with fast kinetics, indicated similar efficiencies in Ca2+-transport. Our results establish PMCA-Neuroplastin complexes as Ca2+-transporters with unanticipated high transport rates and demonstrate that under cellular conditions ATPases may operate in the kHz range.

Endophytic microbes conferring biocontrol are an eco-friendly alternative to control diseases in crops. Unfortunately, the use of endophytes to control diseases is not yet widespread as their application in agricultural settings is challenging and the outcome variable. Translating strains that perform well under laboratory conditions to the field poses several challenges. One is large scale inoculum production in a cost-effective manner. Here, we developed a framework to scale up inoculum production of Fusarium oxysporum 47 (Fo47), assess inoculum viability and its performance in the field and effects on potato yield and performance. The Fo47 endophyte is a well-described biocontrol agent, isolated from disease suppressive soils in the 1980’s. Using mung bean medium, we could routinely produce ≈7 × 108 spores/mL. Using 60 mL of 107 spores/mL per tuber we could re-isolate the fungus 79 days after application from 60 to 70% of the inoculated plants in a large-scale potato field trial (Clenze, Germany). Furthermore, this protocol can be used to assess Fo47 biocontrol potential under field conditions. The presence of the fungus did not negatively affect plant yield or starch production and did not increase susceptibility to endemic pathogens.

The plant immune system employs resistance (R) genes to detect the presence of pathogenic microbes by the avirulence (Avr) factors they produce. Whereas some R-genes confer extreme resistance, completely blocking pathogen proliferation, others act later during infection and allow initial microbial multiplication in the host. We hypothesized that transmembrane R-proteins – as opposed to intracellular R-proteins - may recognize and arrest pathogen entry earlier in the infection process. In tomato, two transmembrane R-proteins (I and I-3) and one intracellular R-protein (I-2) have been identified conferring resistance against the same pathogen: the soil borne fungus Fusarium oxysporum f.sp. lycopersici (Fol). We found that in all root inoculations with Fol on resistant tomato plants, the fungus was able to reach the vasculature of the stem. However, the extent of host vasculature colonization was less than that in susceptible plants. This indicates that a complete blockade of fungal ingress does not occur in any of the incompatible interactions. However, resistance mediated by the intracellular R-protein I-2 allowed more extensive fungal colonization than resistance mediated by the transmembrane R-proteins I or I-3. The first phases of invasion – penetration of the root epidermis, cortex colonization and early stage xylem colonization – are unaffected by R-gene mediated resistance of the host. We suggest that all 3 R-proteins only limit Fol colonization after the fungus has reached the xylem tissues, and that I and I-3 act prior to and/or are more effective than I-2 in reducing pathogen proliferation and spread.

Voltage-activated calcium (Cav) channels couple intracellular signaling pathways to membrane potential by providing Ca2+ ions as second messengers at sufficiently high concentrations to modulate effector proteins located in the intimate vicinity of those channels. Here we show that protein kinase Cβ (PKCβ) and brain nitric oxide synthase (NOS1), both identified by proteomic analysis as constituents of the protein nano-environment of Cav2 channels in the brain, directly coassemble with Cav2.2 channels upon heterologous coexpression. Within Cav2.2-PKCβ and Cav2.2-NOS1 complexes voltage-triggered Ca2+ influx through the Cav channels reliably initiates enzymatic activity within milliseconds. Using BKCa channels as target sensors for nitric oxide and protein phosphorylation together with high concentrations of Ca2+ buffers showed that the complex-mediated Ca2+ signaling occurs in local signaling domains at the plasma membrane. Our results establish Cav2-enzyme complexes as molecular entities for fast electrochemical coupling that reliably convert brief membrane depolarization into precisely timed intracellular signaling events in the mammalian brain.

Infant complementary feeding is important for establishing food preferences. Few studies exist on the effects of infant complementary feeding choices (food preparation methods) on dietary intake, growth or adiposity. We examined whether provision of homemade complementary food is associated with the development of dietary diversity, nutrient intakes and quality of infant growth.Secondary analysis of feeding practices from a randomized trial of vitamin D supplementation in 132 healthy breastfed 1-month-old infants from Montréal, Canada. This longitudinal study used diet records, anthropometric and body composition data (dual-energy X-ray absorptiometry) from assessments that occurred when infants were 6, 9, 12 and 36 months of age. Infants were grouped into three categories of food preparation method on the basis of whether or not they had consumed homemade or commercial meat or fruit and vegetable by 9 months (homemade, commercial and both). Multivariable regression controlled for family income, maternal education and infant sex.Dietary data were available for 65 infants. By 9 months, 22% of infants had exclusively received homemade (n=14), 14 infants had exclusively received commercial and 37 infants had received both. The development of dietary diversity (number of World Health Organization-recommended food groups) was higher (0.76 (95% confidence interval (CI): 0.14, 1.38); P0.05) in the homemade group versus commercial. Energy and nutrient intakes did not differ by group over time. The homemade group had 773 g (-1364, -182; P0.01) lower whole-body fat mass and 7.1% (-12.6, -1.6; P0.05) lower % body fat at 12 months compared with the reference group (both homemade and commercial). Reduced whole-body fat mass in the homemade group persisted at 36 months (-696 g (95% CI: -1341, -52); P0.05). There were no differences between groups for changes in growth Z-scores (length-for-age, weight-for-age and body mass index-for-age).Provision of homemade complementary food is associated with increased dietary diversity during the first year of life and reduced adiposity.

In the original article, there was an error. The amount of xylem sap protein used for nLC-MS/MS analysis was incorrectly depicted; instead of 540 µg of protein 60 µg of protein was TCA precipitated and used for SDS-polyacrylamide gel electrophoresis. A correction has been made to the MATERIALS AND METHODS section, in the sub-section Sample Preparation for nLC-MS/MS: Potential fungal spores were removed from the sap by centrifugation at 800 ×g for 10 min. Xylem sap proteins were concentrated by passing 12 ml of cleared sap through Amicon Ultra-15 Filter Units (Millipore). After centrifugation at 2500 ×g for 15–30 min retentates containing the proteins were recovered. A BCA (bicinchoninic acid) assay (ThermoFischer) was performed to determine the protein concentration. Based on BCA quantification, a volume containing 60 µg of protein was trichloroacetic acid/aceton-precipitated and the pellet was resuspended in SDS loading buffer (2% SDS, 10% glycerol, 60 mM TRIS-HCl pH 6.8, 5% β-mercaptoethanol, 0.01% bromophenol blue), heated at 98◦ C for 5 min and loaded on a 12% SDS-polyacrylamide gel. Following a short electrophoresis, the proteins were stained overnight at 4◦ C with Commassie PageBlue (ThermoFischer). The bands containing the proteins were excised and cysteine reduction and alkylation of the proteins was performed by adding 10 mM DTT pH 8 (incubation at 60◦ C for 1 h) and 20 mM iodoacetamide pH 8 (incubation at room temperature in the dark for 30 min). Protein-containing gel slices were chopped into pieces of approximately 1 mm2 and transferred to 1.5 ml low-binding tubes (Protein LoBind microcentrifuge tubes, Eppendorf). Tryptic in-gel digestion was performed overnight by adding 50 µl of 5 ng/µl Trypsin Sequencing Grade (Sigma-Aldrich). In-house prepared µcolumns were set up by adding C18 Empore disk and LichroprepC18 column material into a 200 µl pipette tip and the tryptic peptides were eluted from the µcolumn with 50 µl of 50% acetonitrile. Acetonitrile content was reduced to

Root endophytes can confer resistance against plant pathogens by direct antagonism or via the host by triggering induced resistance. The latter response typically relies on jasmonic acid (JA)/ethylene (ET)-depended signaling pathways, but can also be triggered via salicylic acid (SA)-dependent signaling pathways. Here, we set out to determine if endophyte-mediated resistance (EMR), conferred by the Fusarium endophyte Fo47, against Fusarium wilt disease in tomato is mediated via SA, ET or JA. To test the contribution of SA, ET, and JA in EMR we performed bioassays with Fo47 and Fusarium oxysporum f. sp. lycopersici in tomato plants impaired in SA accumulation (NahG), JA biosynthesis (def1) or ET-production (ACD) and -sensing (Nr). We observed that the colonization pattern of Fo47 in stems of wildtype plants was indistinguishable from that of the hormone mutants. Surprisingly, EMR was not compromised in the lines affected in JA, ET, or SA signaling. The independence of EMR on SA, JA, and ET implies that this induced resistance-response against Fusarium wilt disease is distinct from the classical Induced Systemic Resistance (ISR) response, providing exciting possibilities for control of wilt diseases independent of conventional defense pathways.

Root colonization by Fusarium oxysporum (Fo) endophytes reduces wilt disease symptoms caused by pathogenic Fo strains. The endophytic strain Fo47, isolated from wilt suppressive soils, reduces Fusarium wilt in various crop species such as tomato, flax, and asparagus. How endophyte-mediated resistance (EMR) against Fusarium wilt is achieved is unclear. Here, nonpathogenic colonization by Fo47 and pathogenic colonization by Fo f.sp. lycopersici (Fol) strains were assessed in tomato roots and stems when inoculated separately or coinoculated. It is shown that Fo47 reduces Fol colonization in stems of both noncultivated and cultivated tomato species. Conversely, Fo47 colonization of coinoculated tomato stems was increased compared to single inoculated plants. Quantitative PCR of fungal colonization of roots (co)inoculated with Fo47 and/or Fol showed that pathogen colonization was drastically reduced when coinoculated with Fo47, compared with single inoculated roots. Endophytic colonization of tomato roots remained unchanged upon coinoculation with Fol. In conclusion, EMR against Fusarium wilt is correlated with a reduction of root and stem colonization by the pathogen. In addition, the endophyte may take advantage of the pathogen-induced suppression of plant defences as it colonizes tomato stems more extensively.

HL-1 cells are currently the only cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining a differentiated cardiac phenotype. Extensive characterization using microscopic, genetic, immunohistochemical, electrophysiological, and pharmacological techniques has demonstrated how similar HL-1 cells are to primary cardiomyocytes. In the few years that HL-1 cells have been available, they have been used in a variety of model systems designed to answer important questions regarding cardiac biology at the cellular and molecular levels. Whereas HL-1 cells have been used to study normal cardiomyocyte function with regard to signaling, electrical, metabolic, and transcriptional regulation, they have also been used to address pathological conditions such as hypoxia, hyperglycemia-hyperinsulinemia, apoptosis, and ischemia-reperfusion. The availability of an immortalized, contractile cardiac cell line has provided investigators with a tool for probing the intricacies of cardiomyocyte function. In this review, we describe the culture and characterization of HL-1 cardiomyocytes as well as various model systems that have been developed using these cells to gain a better understanding of cardiac biology at the cellular and molecular levels.

Plasma membrane Ca2+-ATPases (PMCAs), a family of P-type ATPases, extrude Ca2+ ions from the cytosol to the extracellular space and are considered to be key regulators of Ca2+ signaling. Here we show by functional proteomics that native PMCAs are heteromeric complexes that are assembled from two pore-forming PMCA1-4 subunits and two of the single-span membrane proteins, either neuroplastin or basigin. Contribution of the two Ig domain-containing proteins varies among different types of cells and along postnatal development. Complex formation of neuroplastin or basigin with PMCAs1-4 occurs in the endoplasmic reticulum and is obligatory for stability of the PMCA proteins and for delivery of PMCA complexes to the surface membrane. Knockout and (over)-expression of both neuroplastin and basigin profoundly affect the time course of PMCA-mediated Ca2+ transport, as well as submembraneous Ca2+ concentrations under steady-state conditions. Together, these results establish neuroplastin and basigin as obligatory auxiliary subunits of native PMCAs and key regulators of intracellular Ca2+ concentration.

The stereochemistry of the reaction catalysed by Serratia marcescens chitobiase was determined by HPLC separation of the anomers of N-acetylglucosamine produced during the hydrolysis of p-nitrophenyl N-acetyl-β-D-glucosaminide (PNP-GlcNAc). In the early stages of the reaction, the β-anomer was found to prevail, whereas the α-anomer dominated at mutarotation equilibrium. This established that chitobiase hydrolyses glycosidic bonds with overall retention of the anomeric configuration. Chitobiase-catalysed hydrolysis of PNP-GlcNAc was competitively inhibited by a series of chito-oligosaccharides (degree of polymerization 2-5) that were selectively de-N-acetylated at their non-reducing end. The results are in accord with the participation of the acetamido group at C-2 of the substrate in the catalytic mechanism of chitobiase and related enzymes.

Oncostatin M (OSM) is a member of the interleukin-6 cytokine family and regulates eg. gene activation, cell survival, proliferation and differentiation. OSM binds to a receptor complex consisting of the ubiquitously expressed signal transducer gp130 and the ligand binding OSM receptor subunit, which is expressed on a specific subset of primary afferent neurons. In the present study, the effect of OSM on heat nociception was investigated in nociceptor-specific gp130 knock-out (SNS-gp130-/-) and gp130 floxed (gp130fl/fl) mice. Subcutaneous injection of pathophysiologically relevant concentrations of OSM into the hind-paw of C57BL6J wild type mice significantly reduced paw withdrawal latencies to heat stimulation. In contrast to gp130fl/fl mice, OSM did not induce heat hypersensitivity in vivo in SNS-gp130-/- mice. OSM applied at the receptive fields of sensory neurons in in vitro skin-nerve preparations showed that OSM significantly increased the discharge rate during a standard ramp-shaped heat stimulus. The capsaicin- and heat-sensitive ion channel TRPV1, expressed on a subpopulation of nociceptive neurons, has been shown to play an important role in inflammation-induced heat hypersensitivity. Stimulation of cultured dorsal root ganglion neurons with OSM resulted in potentiation of capsaicin induced ionic currents. In line with these recordings, mice with a null mutation of the TRPV1 gene did not show any signs of OSM-induced heat hypersensitivity in vivo. The present data suggest that OSM induces thermal hypersensitivity by directly sensitizing nociceptors via OSMR-gp130 receptor mediated potentiation of TRPV1.

Peripheral injury or inflammation leads to a release of mediators capable of binding to a variety of ion channels and receptors. Among these are the 7-transmembrane receptors (G protein-coupled receptors) coupling to G(s), G(i/o), G₁₂/₁₃, or G(q/11) G proteins. Each of the G protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of individual signaling pathways in vivo has not yet been worked out. The G(q)/G₁₁ signaling branch is of particular interest because it leads to the activation of phospholipase C-β, protein kinase C, the release of calcium from intracellular stores, and it modulates extracellular regulated kinases. To investigate the contribution of the entire G(q/11)-signaling pathway in nociceptors towards regulation of pain, we generated double-deficient mice lacking G(q/11) selectively in nociceptors using a conditional gene-targeting approach. We observed that nociceptor-specific loss of G(q) and G₁₁ results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. Surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in G(q/11) mutant mice, suggesting a novel function for G(q/11) in tonic modulation of acute nociception. Patch-clamp recordings revealed changes in voltage-dependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of G(q/11), whereas potassium currents remained unchanged. Our results indicate that the functional role of the G(q)/G₁₁ branch of G-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain.

Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P₁ receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P₁ receptor. Our data show that neuronally expressed S1P₁ receptors play a significant role in regulating nociceptor function and that S1P/S1P₁ signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.

This review focuses on the mobility of small RNA (sRNA) molecules from the perspective of trans-kingdom gene silencing. Mobility of sRNA molecules within organisms is a well-known phenomenon, facilitating gene silencing between cells and tissues. sRNA signals are also transmitted between organisms of the same species and of different species. Remarkably, in recent years many examples of RNA-signal exchange have been described to occur between organisms of different kingdoms. These examples are predominantly found in interactions between hosts and their pathogens, parasites, and symbionts. However, they may only represent the tip of the iceberg, since the emerging picture suggests that organisms in biological niches commonly exchange RNA-silencing signals. In this case, we need to take this into account fully to understand how a given biological equilibrium is obtained. Despite many observations of trans-kingdom RNA signal transfer, several mechanistic aspects of these signals remain unknown. Such RNA signal transfer is already being exploited for practical purposes, though. Pathogen genes can be silenced by plant-produced sRNAs designed to affect these genes. This is also known as Host-Induced Genes Silencing (HIGS), and it has the potential to become an important disease-control method in the future.