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Fusaric acid (FA), a picolinic acid derivative, is a natural substance produced by a wide variety of fungal plant pathogens belonging to the Fusarium genus. As a metabolite, fusaric acid exerts several biological activities including metal chelation, electrolyte leakage, repression of ATP synthesis, and direct toxicity on plants, animals and bacteria. Prior studies on the structure of fusaric acid revealed a co-crystal dimeric adduct between FA and 9,10-dehydrofusaric acid. During an ongoing search for signaling genes differentially regulating FA production in the fungal pathogen Fusarium oxysporum ( Fo ), we found that mutants lacking pheromone expression have an increased production of FA compared to the wild type strain. Noteworthy, crystallographic analysis of FA extracted from Fo culture supernatants showed that crystals are formed by a dimeric form of two FA molecules (1:1 molar stoichiometry). Overall, our results suggest that pheromone signaling in Fo is required to regulate the synthesis of fusaric acid.

Soil-inhabiting fungal pathogens use chemical signals released by roots to direct hyphal growth towards the host plant. Whether other soil microorganisms exploit this capacity for their own benefit is currently unknown. Here we show that the endophytic rhizobacterium Rahnella aquatilis locates hyphae of the root-infecting fungal pathogen Fusarium oxysporum through pH-mediated chemotaxis and uses them as highways to efficiently access and colonize plant roots. Secretion of gluconic acid (GlcA) by R. aquatilis in the rhizosphere leads to acidification and counteracts F. oxysporum-induced alkalinisation, a known virulence mechanism, thereby preventing fungal infection. Genetic abrogation or biochemical inhibition of GlcA-mediated acidification abolished biocontrol activity of R. aquatilis and restored fungal infection. These findings reveal a new way by which bacterial endophytes hijack hyphae of a fungal pathogen in the soil to gain preferential access to plant roots, thereby protecting the host from infection.

Autocrine self-signalling via secreted peptides and cognate receptors regulates cell development in eukaryotes and is conserved from protozoans to mammals 1,2 . In contrast, secreted peptides from higher fungi have been traditionally associated with paracrine non-self-signalling during sexual reproduction 3 . For example, cells of the model fungus Saccharomyces cerevisiae fall into two distinct mating types (MAT), which produce either a- or α-pheromone and the cognate receptors Ste2 or Ste3, respectively 4 . Inappropriate autocrine pheromone signalling (APS) during mating is prevented by downregulation of the self-pheromone receptor 4,5 and by a-type cell-specific cleavage of α-pheromone through the protease Bar1 (refs. 6-8 ). While APS can be artificially induced by manipulation of the pheromone secrete-and-sense circuit 7,9-11 , its natural occurrence in ascomycete fungi has not been described. Here, we show that Fusarium oxysporum-a devastating plant pathogen that lacks a known sexual cycle 12 -co-expresses both pheromone-receptor pairs, resulting in autocrine regulation of developmental programmes other than mating. We found that unisexual populations of MAT1-1 cells (α-type idiomorphs 13 ) secrete and sense both a- and α-pheromone, and that their perception requires the cognate receptors and conserved elements of the cell wall integrity mitogen-activated protein kinase cascade. We further show that F. oxysporum uses APS to regulate spore germination in a cell-density-dependent manner, whereby the α-Ste2 interaction leads to repression of conidial germination while the a-Ste3 interaction relieves repression. Our results reveal the existence of a regulatory function for peptide pheromones in the quorum-sensing-mediated control of fungal development.

Plant roots release complex mixtures of bioactive molecules, including compounds that affect the activity and modify the composition of the rhizosphere microbiome. In this work, we investigated the initial phase of the interaction between tomato and an effective biocontrol strain of Trichoderma harzianum (T22). We found that root exudates (RE), obtained from plants grown in a split-root system and exposed to various biotic and abiotic stress factors (wounding, salt, pathogen attack), were able to stimulate the growth and act as chemoattractants of the biocontrol fungus. On the other hand, some of the treatments did not result in an enhanced chemotropism on Fusarium oxysporum f. sp. lycopersici, indicating a mechanism that may be selective for nonpathogenic microbes. The involvement of peroxidases and oxylipins, both known to be released by roots in response to stress, was demonstrated by using RE fractions containing these molecules or their commercial purified analogs, testing the effect of an inhibitor, and characterizing the complex pattern of these metabolites released by tomato roots both locally and systemically.

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 β-turn resembling that of its yeast counterpart. Disruption of the-fold by d-alanine substitution of the conserved central Gly 6 -Gln 7 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 β-turn but instead required two conserved Trp 1 -Cys 2 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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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. [ABSTRACT FROM AUTHOR]