Stability and Qualification of a Legacy Fungal Collection.

Background: Microbial culture collections are valuable repositories for qualified and diverse microorganisms, playing a pivotal role in research, education, innovation, as well as in our response to current and emerging public health and societal challenges. However, such precious holdings, when not integrated in professional biobank infrastructures, may be vulnerable to major risks such as staff retirement, changes in the institutional strategy, or natural disasters. The process of preserving and rescuing "historical" collections can be long and treacherous with a loss of a part of the collection. At the Biological Resource Center of Institut Pasteur, we undertook the challenge of rescuing the dormant legacy fungal collection. Materials and Methods: A total of 64 freeze-dried strains, including yeasts and filamentous fungi, were characterized by using a polyphasic approach combining morphological features and molecular data. We assessed the viability, purity, and authenticity of selected strains isolated from multiple sources and stored for more than 20 years. Results: Our preliminary results show long-term stability of the selected strains and successful qualification in terms of purity and authentication. Moreover, based on the most recent taxonomic revisions, we updated and revised the nomenclature, where applicable. Conclusion: Our findings demonstrated the potential value of reviving historical microbial collections for biobanking and research activities and reassure us about the collection's future reopening.

The Pga59 cell wall protein is an amyloid forming protein involved in adhesion and biofilm establishment in the pathogenic yeast Candida albicans.

The human commensal fungus Candida albicans can attach to epithelia or indwelling medical devices and form biofilms, that are highly tolerant to antifungal drugs and can evade the immune response. The cell surface protein Pga59 has been shown to influence adhesion and biofilm formation. Here, we present evidence that Pga59 displays amyloid properties. Using electron microscopy, staining with an amyloid fibre-specific dye and X-ray diffraction experiments, we showed that the predicted amyloid-forming region of Pga59 is sufficient to build up an amyloid fibre in vitro and that recombinant Pga59 can also adopt a cross-ß amyloid fibre architecture. Further, mutations impairing Pga59 amyloid assembly led to diminished adhesion to substrates and reduced biofilm production. Immunogold labelling on amyloid structures extracted from C. albicans revealed that Pga59 is used by the fungal cell to assemble amyloids within the cell wall in response to adhesion. Altogether, our results suggest that Pga59 amyloid properties are used by the fungal cell to mediate cell-substrate interactions and biofilm formation.

Involvement of amyloid proteins in the formation of biofilms in the pathogenic yeast Candida albicans.

Candida species represent a major fungal threat for human health. Within the Candida genus, the yeast Candida albicans is the most frequently incriminated species during episodes of candidiasis or candidemia. Biofilm formation is used by C. albicans to produce a microbial community that is important in an infectious context. The cell wall, the most superficial cellular compartment, is of paramount importance regarding the establishment of biofilms. C. albicans cell wall contains proteins with amyloid properties that are necessary for biofilm formation due to their adhesion properties. This review focuses on these amyloid proteins during biofilm formation in the yeast C. albicans.

NADPH oxidase regulates chemotropic growth of the fungal pathogen Fusarium oxysporum towards the host plant.

Soil-inhabiting fungal pathogens use chemical signals to locate and colonise the host plant. In the vascular wilt fungus Fusarium oxysporum, hyphal chemotropism towards tomato roots is triggered by secreted plant peroxidases (Prx), which catalyse the reductive cleavage of reactive oxygen species (ROS). Here we show that this chemotropic response requires the regulated synthesis of ROS by the conserved fungal NADPH oxidase B (NoxB) complex, and their transformation into hydrogen peroxide (H2 O2 ) by superoxide dismutase (SOD). Deletion of NoxB or the regulatory subunit NoxR, or pharmacological inhibition of SOD, specifically abolished chemotropism of F. oxysporum towards Prx gradients. Addition of isotropic concentrations of H2 O2 rescued chemotropic growth in the noxBΔ and noxRΔ mutants, but not in a mutant lacking the G protein-coupled receptor Ste2. Prx-triggered rapid Nox- and Ste2-dependent phosphorylation of the cell wall integrity mitogen-activated protein kinase (CWI MAPK) Mpk1, an essential component of the chemotropic response. These results suggest that Ste2 and the CWI MAPK cascade function downstream of NoxB in Prx chemosensing. Our findings reveal a new role for Nox enzymes in directed hyphal growth of a filamentous pathogen towards its host and might be of broad interest for chemotropic interactions between plants and root-colonising fungi.

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.

Hyphal chemotropism in fungal pathogenicity.

The ability to grow as filamentous hyphae defines the lifestyle of fungi. Hyphae are exposed to a variety of chemical stimuli such as nutrients or signal molecules from mating partners and host organisms. How fungi sense and process this chemical information to steer hyphal growth is poorly understood. Saccharomyces cerevisiae and Neurospora crassa have served as genetic models for the identification of cellular components functioning in chemotropism. A recent study in the pathogen Fusarium oxysporum revealed distinct MAPK pathways governing hyphal growth towards nutrient sources and sex pheromones or plant signals, suggesting an unanticipated complexity of chemosensing during fungus-host interactions.

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.

Fungal model systems and the elucidation of pathogenicity determinants.

Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity.

Epigenetic control of effector gene expression in the plant pathogenic fungus Leptosphaeria maculans.

Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions.