Alkaline pH, Low Iron Availability, Poor Nitrogen Sources and CWI MAPK Signaling Are Associated with Increased Fusaric Acid Production in Fusarium oxysporum.

Fusaric acid (FA) is one of the first secondary metabolites isolated from phytopathogenic fungi belonging to the genus Fusarium. This molecule exerts a toxic effect on plants, rhizobacteria, fungi and animals, and it plays a crucial role in both plant and animal pathogenesis. In plants, metal chelation by FA is considered one of the possible mechanisms of action. Here, we evaluated the effect of different nitrogen sources, iron content, extracellular pH and cellular signalling pathways on the production of FA siderophores by the pathogen Fusarium oxysporum (Fol). Our results show that the nitrogen source affects iron chelating activity and FA production. Moreover, alkaline pH and iron limitation boost FA production, while acidic pH and iron sufficiency repress it independent of the nitrogen source. FA production is also positively regulated by the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway and inhibited by the iron homeostasis transcriptional regulator HapX. Collectively, this study demonstrates that factors promoting virulence (i.e., alkaline pH, low iron availability, poor nitrogen sources and CWI MAPK signalling) are also associated with increased FA production in Fol. The obtained new insights on FA biosynthesis regulation can be used to prevent both Fol infection potential and toxin contamination.

Localisation of phosphoinositides in the grass endophyte Epichloë festucae and genetic and functional analysis of key components of their biosynthetic pathway in E. festucae symbiosis and Fusarium oxysporum pathogenesis.

Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signaling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localize the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloë festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homolog of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did enable the detection of PI[3,4,5]P3 in septa and mycelium of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P3 in ΔtepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 and that fungal PTEN homologs are functional lipid phosphatases. The F. oxysporum chemotropism defect suggests a conserved role of PTEN homologs in chemotaxis across protists, fungi and mammals.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Structure of Fungal α Mating Pheromone in Membrane Mimetics Suggests a Possible Role for Regulation at the Water-Membrane Interface.

Fusarium oxysporum is a highly destructive plant pathogen and an emerging pathogen of humans. Like other ascomycete fungi, F. oxysporum secretes α-pheromone, a small peptide that functions both as a chemoattractant and as a quorum-sensing signal. Three of the ten amino acid residues of α-pheromone are tryptophan, an amino acid whose sidechain has high affinity for lipid bilayers, suggesting a possible interaction with biological membranes. Here we tested the effect of different lipid environments on α-pheromone structure and function. Using spectroscopic and calorimetric approaches, we show that this peptide interacts with negatively charged model phospholipid vesicles. Fluorescence emission spectroscopy and nuclear magnetic resonance (NMR) measurements revealed a key role of the positively charged groups and Trp residues. Furthermore, NMR-based calculation of the 3D structure in the presence of micelles, formed by lipid surfactants, suggests that α-pheromone can establish an intramolecular disulfide bond between the two cysteine residues during interaction with membranes, but not in the absence of lipid mimetics. Remarkably, this oxidized version of α-pheromone lacks biological activity as a chemoattractant and quorum-sensing molecule. These results suggest the presence of a previously unidentified redox regulated control of α-pheromone activity at the surface of the plasma membrane that could influence the interaction with its cognate G-protein coupled receptor.

The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set of lineage-specific chromosomes.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation.

A Conserved Microbial Motif 'Traps' Protease Activation in Host Immunity.

A recent study (Misas-Villamil et al., Nat. Commun., 2019) reveals that Pit2, an apoplastic effector of the corn smut fungus Ustilago maydis, contains an embedded motif of 14 amino acids that binds to and inhibits plant cysteine proteases, thereby modulating host immunity. Intriguingly, the inhibitory motif acts by mimicking the protease substrate and is conserved across microbial kingdoms.

Characterization of a novel cysteine-rich antifungal protein from Fusarium graminearum with activity against maize fungal pathogens.

Filamentous fungi are an invaluable source for biocontrol strategies and for production and development of different antifungal polypeptides. Within this context, cysteine-rich antifungal AFP-like peptides stand out among many different antimicrobial compounds given their production easiness, stability, versatility, and efficacy. AFP from Aspergillus giganteus represents the hallmark of this still increasing family of antifungal polypeptides. Close in silico analyses of the Fusarium graminearum genome revealed the presence of an AFP-like peptide, here designated as FgAFP. This new peptide was cloned, produced in the yeast Pichia pastoris, and characterized. The results obtained showed its strong and specific antifungal activity against several well-recognized maize pathogens, but inefficacy against F. oxysporum, which has not been described as a natural biological competitor of other fungal pathogens assayed. All results together suggest that this small peptide is an important factor for the fungal interplays involved in maize infection and reveals unforeseen potential biotechnological applications for FgAFP in maize production and storage.

Histomorphologic and ultrastructural recovery of myopathy in rats treated with low-level laser therapy.

The purpose of the present work was to study the effect of low-level laser therapy (LLLT): helium-neon (He-Ne) and gallium arsenide (Ga-As) laser on the histomorphology of muscle and mitochondria in experimental myopathy in rats. Thirty Suquía strain female rats were distributed in groups: (A) control (intact), (B) injured, (C) injured and treated with He-Ne laser, (D) injured and treated with Ga-As laser, (E) irradiated with He-Ne laser on the non-injured muscle, and (F) irradiated with Ga-As laser on the non-injured muscle. Myopathy was induced by injecting 0.05 mg/rat/day of adrenaline in the left gastrocnemius muscle at the same point on five consecutive days, in groups B, C, and D. LLLT was applied with 9.5 J cm-2 daily for seven consecutive days in groups C, D, E, and F. The muscles were examined with optic and electronic microscopy. The inflammation was classified as absent, mild, and intense and the degree of mitochondrial alteration was graded I, II, III, and IV. Categorical data were statistically analyzed by Chi-square and the Fisher-Irwin Bilateral test, setting significant difference at p < 0.05. The damage found in muscle and mitochondria histomorphology in animals with induced myopathy (B) was intense or severe inflammation with grade III or IV of mitochondrial alteration. They underwent significant regression (p < 0.001) compared with the groups treated with He-Ne (C) and Ga-As (D) laser, in which mild or moderate inflammation was seen and mitochondrial alteration grades I and II, recovering normal myofibrillar architecture. No differences were found between the effects caused by the two lasers, or between groups A, E, and F. Group A was found to be different from B, C, and D (p < 0.001). LLLT in experimental myopathy caused significant muscular and mitochondrial morphologic recovery.

Structure-Activity Relationship of α Mating Pheromone from the Fungal Pathogen Fusarium oxysporum.

During sexual development ascomycete fungi produce two types of peptide pheromones termed a and α. The α pheromone from the budding yeast Saccharomyces cerevisiae, a 13-residue peptide that elicits cell cycle arrest and chemotropic growth, has served as paradigm for the interaction of small peptides with their cognate G protein-coupled receptors. However, no structural information is currently available for α pheromones from filamentous ascomycetes, which are significantly shorter and share almost no sequence similarity with the S. cerevisiae homolog. High resolution structure of synthetic α-pheromone from the plant pathogenic ascomycete Fusarium oxysporum revealed the presence of a central ß-turn resembling that of its yeast counterpart. Disruption of the-fold by d-alanine substitution of the conserved central Gly6-Gln7 residues or by random sequence scrambling demonstrated a crucial role for this structural determinant in chemoattractant activity. Unexpectedly, the growth inhibitory effect of F. oxysporum α-pheromone was independent of the cognate G protein-coupled receptors Ste2 and of the central ß-turn but instead required two conserved Trp1-Cys2 residues at the N terminus. These results indicate that, despite their reduced size, fungal α-pheromones contain discrete functional regions with a defined secondary structure that regulate diverse biological processes such as polarity reorientation and cell division.

Plant Defensins NaD1 and NaD2 Induce Different Stress Response Pathways in Fungi.

Nicotiana alata defensins 1 and 2 (NaD1 and NaD2) are plant defensins from the ornamental tobacco that have antifungal activity against a variety of fungal pathogens. Some plant defensins interact with fungal cell wall O-glycosylated proteins. Therefore, we investigated if this was the case for NaD1 and NaD2, by assessing the sensitivity of the three Aspergillus nidulans (An) O-mannosyltransferase (pmt) knockout (KO) mutants (An∆pmtA, An∆pmtB, and An∆pmtC). An∆pmtA was resistant to both defensins, while An∆pmtC was resistant to NaD2 only, suggesting NaD1 and NaD2 are unlikely to have a general interaction with O-linked side chains. Further evidence of this difference in the antifungal mechanism was provided by the dissimilarity of the NaD1 and NaD2 sensitivities of the Fusarium oxysporum f. sp. lycopersici (Fol) signalling knockout mutants from the cell wall integrity (CWI) and high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathways. HOG pathway mutants were sensitive to both NaD1 and NaD2, while CWI pathway mutants only displayed sensitivity to NaD2.

A fungal pathogen secretes plant alkalinizing peptides to increase infection.

Plant infections caused by fungi are often associated with an increase in the pH of the surrounding host tissue(1). Extracellular alkalinization is thought to contribute to fungal pathogenesis, but the underlying mechanisms are poorly understood. Here, we show that the root-infecting fungus Fusarium oxysporum uses a functional homologue of the plant regulatory peptide RALF (rapid alkalinization factor)(2,3) to induce alkalinization and cause disease in plants. An upshift in extracellular pH promotes infectious growth of Fusarium by stimulating phosphorylation of a conserved mitogen-activated protein kinase essential for pathogenicity(4,5). Fungal mutants lacking a functional Fusarium (F)-RALF peptide failed to induce host alkalinization and showed markedly reduced virulence in tomato plants, while eliciting a strong host immune response. Arabidopsis plants lacking the receptor-like kinase FERONIA, which mediates the RALF-triggered alkalinization response(6), displayed enhanced resistance against Fusarium. RALF homologues are found across a number of phylogenetically distant groups of fungi, many of which infect plants. We propose that fungal pathogens use functional homologues of alkalinizing peptides found in their host plants to increase their infectious potential and suppress host immunity.

Fungal pathogen uses sex pheromone receptor for chemotropic sensing of host plant signals.

For more than a century, fungal pathogens and symbionts have been known to orient hyphal growth towards chemical stimuli from the host plant. However, the nature of the plant signals as well as the mechanisms underlying the chemotropic response have remained elusive. Here we show that directed growth of the soil-inhabiting plant pathogen Fusarium oxysporum towards the roots of the host tomato (Solanum lycopersicum) is triggered by the catalytic activity of secreted class III peroxidases, a family of haem-containing enzymes present in all land plants. The chemotropic response requires conserved elements of the fungal cell integrity mitogen-activated protein kinase (MAPK) cascade and the seven-pass transmembrane protein Ste2, a functional homologue of the Saccharomyces cerevisiae sex pheromone α receptor. We further show that directed hyphal growth of F. oxysporum towards nutrient sources such as sugars and amino acids is governed by a functionally distinct MAPK cascade. These results reveal a potentially conserved chemotropic mechanism in root-colonizing fungi, and suggest a new function for the fungal pheromone-sensing machinery in locating plant hosts in a complex environment such as the soil.

Protein kinases in plant-pathogenic fungi: conserved regulators of infection.

Phytopathogenic fungi have evolved an amazing diversity of infection modes and nutritional strategies, yet the signaling pathways that govern pathogenicity are remarkably conserved. Protein kinases (PKs) catalyze the reversible phosphorylation of proteins, regulating a variety of cellular processes. Here, we present an overview of our current understanding of the different classes of PKs that contribute to fungal pathogenicity on plants and of the mechanisms that regulate and coordinate PK activity during infection-related development. In addition to the well-studied PK modules, such as MAPK (mitogen-activated protein kinase) and cAMP (cyclic adenosine monophosphate)-PKA (protein kinase A) cascades, we also discuss new PK pathways that have emerged in recent years as key players of pathogenic development and disease. Understanding how conserved PK signaling networks have been recruited during the evolution of fungal pathogenicity not only advances our knowledge of the highly elaborate infection process but may also lead to the development of novel strategies for the control of plant disease.

Hyphal growth of phagocytosed Fusarium oxysporum causes cell lysis and death of murine macrophages.

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.

Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus and Fusarium oxysporum.

Filamentous fungi are an important cause of pulmonary and systemic morbidity and mortality, and also cause corneal blindness and visual impairment worldwide. Utilizing in vitro neutrophil killing assays and a model of fungal infection of the cornea, we demonstrated that Dectin-1 dependent IL-6 production regulates expression of iron chelators, heme and siderophore binding proteins and hepcidin in infected mice. In addition, we show that human neutrophils synthesize lipocalin-1, which sequesters fungal siderophores, and that topical lipocalin-1 or lactoferrin restricts fungal growth in vivo. Conversely, we show that exogenous iron or the xenosiderophore deferroxamine enhances fungal growth in infected mice. By examining mutant Aspergillus and Fusarium strains, we found that fungal transcriptional responses to low iron levels and extracellular siderophores are essential for fungal growth during infection. Further, we showed that targeting fungal iron acquisition or siderophore biosynthesis by topical application of iron chelators or statins reduces fungal growth in the cornea by 60% and that dual therapy with the iron chelator deferiprone and statins further restricts fungal growth by 75%. Together, these studies identify specific host iron-chelating and fungal iron-acquisition mediators that regulate fungal growth, and demonstrate that therapeutic inhibition of fungal iron acquisition can be utilized to treat topical fungal infections.

Iron competition in fungus-plant interactions: the battle takes place in the rhizosphere.

Soilborne fungal pathogens are highly persistent and provoke important crop losses. During saprophytic and infectious stages in the soil, these organisms face situations of nutrient limitation and lack of essential elements, such as iron. We investigated the role of the bZIP transcription factor HapX as a central regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. This root-infecting plant pathogen attacks more than hundred different crops and is an emerging human opportunistic invader. Although iron uptake remains unaffected in a strain lacking HapX, de-repression of genes implicated in iron-consuming processes such as respiration, amino acid metabolism, TCA cycle and heme biosynthesis lead to severely impaired growth under iron-limiting conditions. HapX is required for full virulence of F. oxysporum in tomato plants and essential for infection in immunodepressed mice. Virulence attenuation of the ΔhapX strain on tomato plants is more pronounced by co-inoculation of roots with the biocontrol strain Pseudomonas putida KT2440, but not with a mutant deficient in siderophores production. These results demonstrate that HapX is required for iron competition of F. oxysporum in the tomato rhizosphere and establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals.

The CAP protein superfamily: function in sterol export and fungal virulence.

CAP superfamily proteins, also known as sperm-coating proteins, are found in all kingdoms of life and have been implicated in a variety of physiological contexts, including immune defense in plants and mammals, sperm maturation and fertilization, fungal virulence, and toxicity of insect and reptile venoms as well as prostate and brain cancer. CAP family members are mostly secreted glycoproteins that are highly stable in the extracellular fluid. All members of the superfamily share a common CAP domain of approximately 150 amino acids, which adopts a unique α-ß-α sandwich fold. The conserved structure suggests that CAP proteins exert fundamentally similar functions. However, the molecular mode of action of this protein family has remained enigmatic. The budding yeast Saccharomyces cerevisiae has three CAP family members designated Pry (pathogen related in yeast), and recent evidence indicates that they act as sterol-binding and export proteins. Expression of the mammalian CAP protein CRISP2, which binds sterols in vitro, complements the sterol export defect of a yeast pry mutant, suggesting that sterol binding and export is conserved among different CAP family members. Collectively, these observations suggest that CAP family members constitute a novel class of secreted extracellular sterol-binding proteins. A ligand-binding activity of the CAP domain could explain many of the biological activities attributed to these proteins. For example, the strong induction of plant pathogenesis-related 1 protein upon exposure to pathogens may serve to inhibit pathogen proliferation by extracting sterols from the pathogen membrane. Similarly, the presence of these proteins in the venom of toxic insects and reptiles or in the secretome of pathogenic fungi might inflict damage by sequestering sterols or related small hydrophobic compounds from the host tissue.

Resultados del tratamiento kinésico en un caso de doble parálisis facial / Physiotherapy treatment results in a case of double facial paralysis

Paciente de sexo masculino de 33 años que ingresa al Servicio Universitario de Kinesiología, con diagnóstico de parálisisfacial derecha, por lo cual se inició el tratamiento kinésico con corriente de tipo exponencial, (indicada paratratar músculos que tienen afectada la placa neuromuscular ya que son músculos patológicos), ejercicios de reeducación de la mímica y masoterapia.A los veinte días de evolución, se instala otra parálisis facial del lado izquierdo, cuando aún estaba realizando la rehabilitación de la hemicara derecha, por lo cual se efectuó el tratamiento de manera simultánea, pero con diferenciasen la utilización de la corriente exponencial, variando el ancho de pulso y la intensidad. El trofismo, las unciones, la simetría y la mímica comprometidas por la parálisis facial derecha, fueron recuperadas en 19 sesiones y la izquierda en 15 sesiones, lográndose la simetría total de la cara, sin secuelas perceptibles al final del tratamiento...

Male patient, 33 years old, who enters at the University Department of Kinesiology, diagnosed with right facial paralysis. He was initiating treatment with current exponential type, which is indicated to treat muscles that have affected the neuromuscular junction (muscle disease), exercise rehabilitation of mime and massage therapy. After twenty days of evolution, installs another left–sided facial paralysis, while still doing the right hemifacial rehabilitation, so treatment was performed simultaneously, but with differences in the use of the exponential current, varying the pulse width and intensity. The nutrition, functions, symmetry and mime committed by right facial palsy. were recovered in 19 sessions, and the left after fifteen sessions on achieving the overall symmetry of the face, withoutsqueale noticeable at the end of treatment...

HapX-mediated iron homeostasis is essential for rhizosphere competence and virulence of the soilborne pathogen Fusarium oxysporum.

Soilborne fungal pathogens cause devastating yield losses and are highly persistent and difficult to control. During the infection process, these organisms must cope with limited availability of iron. Here we show that the bZIP protein HapX functions as a key regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. Deletion of hapX does not affect iron uptake but causes derepression of genes involved in iron-consuming pathways, leading to impaired growth under iron-depleted conditions. F. oxysporum strains lacking HapX are reduced in their capacity to invade and kill tomato (Solanum lycopersicum) plants and immunodepressed mice. The virulence defect of ΔhapX on tomato plants is exacerbated by coinoculation of roots with a biocontrol strain of Pseudomonas putida, but not with a siderophore-deficient mutant, indicating that HapX contributes to iron competition of F. oxysporum in the tomato rhizosphere. These results establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.