Location and timing govern tripartite interactions of fungal phytopathogens and host in the stem canker species complex.

BACKGROUND: Leptosphaeria maculans "brassicae" (Lmb) and Leptosphaeria biglobosa "brassicae" (Lbb) make up a species complex involved in the stem canker (blackleg) disease of rapeseed (Brassica napus). They coinfect rapeseed together, from the early stage of infection on leaves to the final necrotic stage at the stem base, and both perform sexual crossings on plant residues. L. biglobosa is suggested to be a potential biocontrol agent against Lmb, but there has been no mechanistic investigation of the different types of interactions that may occur between the plant and the two fungal species. RESULTS: We investigated the bi- or tripartite interaction mechanisms by (i) confronting Lmb and Lbb in culture conditions or during cotyledon infection, with different timing and/or spore concentration regimes, (ii) performing RNA-Seq experiments in vitro or on the kinetics of infection of cotyledons infected by Lmb and/or Lbb to evaluate the transcriptomic activity and the plant response when both fungal species are inoculated together. Lbb infection of B. napus cotyledons was typical of a necrotrophic behavior, with a very early setup of one pathogenicity program and very limited colonization of tissues. This contrasted with the complex succession of pathogenicity programs of the hemibiotroph Lmb. During simultaneous co-infection by both species, Lmb was strongly impacted in its growth and transcriptomic dynamics both in vitro and in planta, while Lbb was unaffected by the presence of Lmb. However, the drastic inhibition of Lmb growth by Lbb was ineffective in the case of delayed inoculation with Lbb or a lower amount of spores of Lbb compared to Lmb. CONCLUSIONS: Our data suggest that Lmb growth inhibition by Lbb is the result of a combination of factors that may include competition for trophic resources, the generation by Lbb of an environment unsuitable for the lifecycle of Lmb or/and the effect on Lmb of plant defense responses induced by Lbb. It indicates that growth inhibition occurs in very specific conditions (i.e., co-inoculation at the same place of an equal amount of inoculum) that are unlikely to occur in the field where their coexistence does not prevent any species from completing their life cycle.

Vaccine development for pathogenic fungi: current status and future directions.

INTRODUCTION: Fungal infections are caused by a broad range of pathogenic fungi that are found worldwide with different geographic distributions, incidences, and mortality rates. Considering that there are relatively few approved medications available for combating fungal diseases and no vaccine formulation commercially available, multiple groups are searching for new antifungal drugs, examining drugs for repurposing and developing antifungal vaccines, in order to control deaths, sequels, and the spread of these complex infections. AREAS COVERED: This review provides a summary of advances in fungal vaccine studies and the different approaches under development, such as subunit vaccines, whole organism vaccines, and DNA vaccines, as well as studies that optimize the use of adjuvants. We conducted a literature search of the PubMed with terms: fungal vaccines and genus of fungal pathogens (Cryptococcus spp. Candida spp. Coccidioides spp. Aspergillus spp. Sporothrix spp. Histoplasma spp. Paracoccidioides spp. Pneumocystis spp. and the Mucorales order), a total of 177 articles were collected from database. EXPERT OPINION: Problems regarding the immune response development in an immunocompromised organism, the similarity between fungal and mammalian cells, and the lack of attention by health organizations to fungal infections are closely related to the fact that, at present, there are no fungal vaccines available for clinical use.

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum.

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

Fungi-on-a-Chip: microfluidic platforms for single-cell studies on fungi.

This review highlights new advances in the emerging field of 'Fungi-on-a-Chip' microfluidics for single-cell studies on fungi and discusses several future frontiers, where we envisage microfluidic technology development to be instrumental in aiding our understanding of fungal biology. Fungi, with their enormous diversity, bear essential roles both in nature and our everyday lives. They inhabit a range of ecosystems, such as soil, where they are involved in organic matter degradation and bioremediation processes. More recently, fungi have been recognized as key components of the microbiome in other eukaryotes, such as humans, where they play a fundamental role not only in human pathogenesis, but also likely as commensals. In the food sector, fungi are used either directly or as fermenting agents and are often key players in the biotechnological industry, where they are responsible for the production of both bulk chemicals and antibiotics. Although the macroscopic fruiting bodies are immediately recognizable by most observers, the structure, function, and interactions of fungi with other microbes at the microscopic scale still remain largely hidden. Herein, we shed light on new advances in the emerging field of Fungi-on-a-Chip microfluidic technologies for single-cell studies on fungi. We discuss the development and application of microfluidic tools in the fields of medicine and biotechnology, as well as in-depth biological studies having significance for ecology and general natural processes. Finally, a future perspective is provided, highlighting new frontiers in which microfluidic technology can benefit this field.

Deletion of AA9 Lytic Polysaccharide Monooxygenases Impacts A. nidulans Secretome and Growth on Lignocellulose.

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes found in viruses, archaea, and bacteria as well as eukaryotes, such as fungi, algae and insects, actively contributing to the degradation of different polysaccharides. In Aspergillus nidulans, LPMOs from family AA9 (AnLPMO9s), along with an AA3 cellobiose dehydrogenase (AnCDH1), are cosecreted upon growth on crystalline cellulose and lignocellulosic substrates, indicating their role in the degradation of plant cell wall components. Functional analysis revealed that three target LPMO9s (AnLPMO9C, AnLPMO9F and AnLPMO9G) correspond to cellulose-active enzymes with distinct regioselectivity and activity on cellulose with different proportions of crystalline and amorphous regions. AnLPMO9s deletion and overexpression studies corroborate functional data. The abundantly secreted AnLPMO9F is a major component of the extracellular cellulolytic system, while AnLPMO9G was less abundant and constantly secreted, and acts preferentially on crystalline regions of cellulose, uniquely displaying activity on highly crystalline algae cellulose. Single or double deletion of AnLPMO9s resulted in about 25% reduction in fungal growth on sugarcane straw but not on Avicel, demonstrating the contribution of LPMO9s for the saprophytic fungal lifestyle relies on the degradation of complex lignocellulosic substrates. Although the deletion of AnCDH1 slightly reduced the cellulolytic activity, it did not affect fungal growth indicating the existence of alternative electron donors to LPMOs. Additionally, double or triple knockouts of these enzymes had no accumulative deleterious effect on the cellulolytic activity nor on fungal growth, regardless of the deleted gene. Overexpression of AnLPMO9s in a cellulose-induced secretome background confirmed the importance and applicability of AnLPMO9G to improve lignocellulose saccharification. IMPORTANCE Fungal lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that boost plant biomass degradation in combination with glycoside hydrolases. Secretion of LPMO9s arsenal by Aspergillus nidulans is influenced by the substrate and time of induction. These findings along with the biochemical characterization of novel fungal LPMO9s have implications on our understanding of their concerted action, allowing rational engineering of fungal strains for biotechnological applications such as plant biomass degradation. Additionally, the role of oxidative players in fungal growth on plant biomass was evaluated by deletion and overexpression experiments using a model fungal system.

Fungal Extracellular Vesicles Are Involved in Intraspecies Intracellular Communication.

Fungal infections are associated with high mortality rates in humans. The risk of fungal diseases creates the urgent need to broaden the knowledge base regarding their pathophysiology. In this sense, the role of extracellular vesicles (EVs) has been described to convey biological information and participate in the fungus-host interaction process. We hypothesized that fungal EVs work as an additional element in the communication routes regulating fungal responses in intraspecies interaction systems. In this respect, the aim of this study was to address the gene regulation profiles prompted by fungal EVs in intraspecies recipient cells. Our data demonstrated the intraspecies uptake of EVs in pathogenic fungi, such as Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis, and the effects triggered by EVs in fungal cells. In C. albicans, we evaluated the involvement of EVs in the yeast-to-hypha transition, while in P. brasiliensis and A. fumigatus the function of EVs as stress transducers was investigated. P. brasiliensis and A. fumigatus were exposed to an inhibitor of glycosylation or UV light, respectively. The results demonstrated the role of EVs in regulating the expression of target genes and triggering phenotypic changes. The EVs treatment induced cellular proliferation and boosted the yeast to hyphal transition in C. albicans, while they enhanced stress responsiveness in A. fumigatus and P. brasiliensis, establishing a role for EVs in fungal intraspecies communication. Thus, EVs regulate fungal behavior, acting as potent message effectors, and understanding their effects and mechanism(s) of action could be exploited in antifungal therapies. IMPORTANCE Here, we report a study about extracellular vesicles (EVs) as communication mediators in fungi. Our results demonstrated the role of EVs from Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis regulating the expression of target genes and phenotypic features. We asked whether fungal EVs play a role as message effectors. We show that fungal EVs are involved in fungal interaction systems as potent message effectors, and understanding their effects and mechanisms of action could be exploited in antifungal therapies.

Perception of fungi by farmers in the Cerrado / Percepção de agricultores do Cerrado sobre os fungos

Fungi are important in several aspects of human life. In particular, to agriculture, pathogenic fungi are of great importance, as they are responsible for production losses of the most diverse types. Because of this, knowledge about pathogenic fungus is of extreme importance for farmers and professionals working in agricultural areas. Among farmers who use specific agroecological practices, this knowledge is even more valuable, since by not adopting conventional methods of production, they resort to non-invasive alternatives that are less or not harmful at all to the environment in consideration of production management methods. This study aimed to assess farmer perception in the Cerrado biome in the city of Goiás (GO), Brazil, in order to understand their ethnomycological perceptions to verify historical management practices, their knowledge about phytopathogenic fungi, and how these producers perceive fungi. We used the theoretical reference method "From peasant to peasant" formulated by ANPA - National Association of Small Farmers. Some aspects of farmers' ethnomycological knowledge are discussed. These ease identification the representatives of the Fungi Kingdom is associated with organisms that present easily recognizable characteristics, such as wood-ears or disease-causing fungi. In general, farmers are able to identify representatives of the Fungi Kingdom that are found in their daily lives. The perception of farmers about fungi, a group still much unknown by society, is very relevant for future actions of ethnomycology.

Percepção dos fungos por agricultores do Cerrado ­ Fungos são organismos importantes em vários aspectos da vida humana. Em particular, para a agricultura, fungos patogênicos são de grande importância, pois são responsáveis por perdas de produção dos mais diversos tipos. Por isso, o conhecimento sobre fungos patogênicos é de extrema importância para agricultores e profissionais que trabalham em áreas agrícolas. Entre os agricultores que utilizam práticas agroecológicas, esse conhecimento é ainda mais valioso, pois, ao não adotar métodos convencionais de produção, recorrem a alternativas não invasivas que são menos ou não prejudiciais ao meio ambiente, considerando os métodos de gerenciamento da produção. Este estudo teve como objetivo avaliar a percepção de um grupo de agricultores do bioma Cerrado, na cidade de Goiás (GO), Brasil, a fim de compreender suas percepções etnomicológicas, de modo a verificar práticas históricas de manejo, seu conhecimento sobre fungos fitopatogênicos e como esses produtores percebem os fungos, de modo geral. Utilizamos o método "de camponês para camponês", formulado pela ANPA ­ Associação Nacional de Pequenos Agricultores. Discutimos alguns aspectos do conhecimento etnomicológico destes agricultores. A facilidade de identificação de representantes fúngicos está associada a organismos que apresentam características facilmente reconhecíveis, como estereótipo orelha-de-pau ou fungos causadores de doenças em cultivares. Em geral, os agricultores do estudo são capazes de identificar representantes do Reino Fungi que são encontrados em suas vidas cotidianas. A percepção dos agricultores sobre os fungos, um grupo ainda muito desconhecido pela sociedade, é muito relevante para ações futuras dentro da etnomicologia.

Perception of fungi by farmers in the Cerrado / Percepção de agricultores do Cerrado sobre os fungos

Fungi are important in several aspects of human life. In particular, to agriculture, pathogenic fungi are of great importance, as they are responsible for production losses of the most diverse types. Because of this, knowledge about pathogenic fungus is of extreme importance for farmers and professionals working in agricultural areas. Among farmers who use specific agroecological practices, this knowledge is even more valuable, since by not adopting conventional methods of production, they resort to non-invasive alternatives that are less or not harmful at all to the environment in consideration of production management methods. This study aimed to assess farmer perception in the Cerrado biome in the city of Goiás (GO), Brazil, in order to understand their ethnomycological perceptions to verify historical management practices, their knowledge about phytopathogenic fungi, and how these producers perceive fungi. We used the theoretical reference method "From peasant to peasant" formulated by ANPA - National Association of Small Farmers. Some aspects of farmers' ethnomycological knowledge are discussed. These ease identification the representatives of the Fungi Kingdom is associated with organisms that present easily recognizable characteristics, such as wood-ears or disease-causing fungi. In general, farmers are able to identify representatives of the Fungi Kingdom that are found in their daily lives. The perception of farmers about fungi, a group still much unknown by society, is very relevant for future actions of ethnomycology.

Percepção dos fungos por agricultores do Cerrado – Fungos são organismos importantes em vários aspectos da vida humana. Em particular, para a agricultura, fungos patogênicos são de grande importância, pois são responsáveis por perdas de produção dos mais diversos tipos. Por isso, o conhecimento sobre fungos patogênicos é de extrema importância para agricultores e profissionais que trabalham em áreas agrícolas. Entre os agricultores que utilizam práticas agroecológicas, esse conhecimento é ainda mais valioso, pois, ao não adotar métodos convencionais de produção, recorrem a alternativas não invasivas que são menos ou não prejudiciais ao meio ambiente, considerando os métodos de gerenciamento da produção. Este estudo teve como objetivo avaliar a percepção de um grupo de agricultores do bioma Cerrado, na cidade de Goiás (GO), Brasil, a fim de compreender suas percepções etnomicológicas, de modo a verificar práticas históricas de manejo, seu conhecimento sobre fungos fitopatogênicos e como esses produtores percebem os fungos, de modo geral. Utilizamos o método "de camponês para camponês", formulado pela ANPA – Associação Nacional de Pequenos Agricultores. Discutimos alguns aspectos do conhecimento etnomicológico destes agricultores. A facilidade de identificação de representantes fúngicos está associada a organismos que apresentam características facilmente reconhecíveis, como estereótipo orelha-de-pau ou fungos causadores de doenças em cultivares. Em geral, os agricultores do estudo são capazes de identificar representantes do Reino Fungi que são encontrados em suas vidas cotidianas. A percepção dos agricultores sobre os fungos, um grupo ainda muito desconhecido pela sociedade, é muito relevante para ações futuras dentro da etnomicologia.

Perception of fungi by farmers in the Cerrado

Abstract Fungi are important in several aspects of human life. In particular, to agriculture, pathogenic fungi are of great importance, as they are responsible for production losses of the most diverse types. Because of this, knowledge about pathogenic fungus is of extreme importance for farmers and professionals working in agricultural areas. Among farmers who use specific agroecological practices, this knowledge is even more valuable, since by not adopting conventional methods of production, they resort to non-invasive alternatives that are less or not harmful at all to the environment in consideration of production management methods. This study aimed to assess farmer perception in the Cerrado biome in the city of Goiás (GO), Brazil, in order to understand their ethnomycological perceptions to verify historical management practices, their knowledge about phytopathogenic fungi, and how these producers perceive fungi. We used the theoretical reference method From peasant to peasant formulated by ANPA - National Association of Small Farmers. Some aspects of farmers ethnomycological knowledge are discussed. These ease identification the representatives of the Fungi Kingdom is associated with organisms that present easily recognizable characteristics, such as wood-ears or disease-causing fungi. In general, farmers are able to identify representatives of the Fungi Kingdom that are found in their daily lives. The perception of farmers about fungi, a group still much unknown by society, is very relevant for future actions of ethnomycology.

Resumo Percepção dos fungos por agricultores do Cerrado Fungos são organismos importantes em vários aspectos da vida humana. Em particular, para a agricultura, fungos patogênicos são de grande importância, pois são responsáveis por perdas de produção dos mais diversos tipos. Por isso, o conhecimento sobre fungos patogênicos é de extrema importância para agricultores e profissionais que trabalham em áreas agrícolas. Entre os agricultores que utilizam práticas agroecológicas, esse conhecimento é ainda mais valioso, pois, ao não adotar métodos convencionais de produção, recorrem a alternativas não invasivas que são menos ou não prejudiciais ao meio ambiente, considerando os métodos de gerenciamento da produção. Este estudo teve como objetivo avaliar a percepção de um grupo de agricultores do bioma Cerrado, na cidade de Goiás (GO), Brasil, a fim de compreender suas percepções etnomicológicas, de modo a verificar práticas históricas de manejo, seu conhecimento sobre fungos fitopatogênicos e como esses produtores percebem os fungos, de modo geral. Utilizamos o método de camponês para camponês, formulado pela ANPA Associação Nacional de Pequenos Agricultores. Discutimos alguns aspectos do conhecimento etnomicológico destes agricultores. A facilidade de identificação de representantes fúngicos está associada a organismos que apresentam características facilmente reconhecíveis, como estereótipo orelha-de-pau ou fungos causadores de doenças em cultivares. Em geral, os agricultores do estudo são capazes de identificar representantes do Reino Fungi que são encontrados em suas vidas cotidianas. A percepção dos agricultores sobre os fungos, um grupo ainda muito desconhecido pela sociedade, é muito relevante para ações futuras dentro da etnomicologia.

Candida albicans Isolates 529L and CHN1 Exhibit Stable Colonization of the Murine Gastrointestinal Tract.

Candida albicans is a pathobiont that colonizes multiple niches in the body including the gastrointestinal (GI) tract but is also responsible for both mucosal and systemic infections. Despite its prevalence as a human commensal, the murine GI tract is generally refractory to colonization with the C. albicans reference isolate SC5314. Here, we identify two C. albicans isolates, 529L and CHN1, that stably colonize the murine GI tract in three different animal facilities under conditions where SC5314 is lost from this niche. Analysis of the bacterial microbiota did not show notable differences among mice colonized with the three C. albicans strains. We compared the genotypes and phenotypes of these three strains and identified thousands of single nucleotide polymorphisms (SNPs) and multiple phenotypic differences, including their ability to grow and filament in response to nutritional cues. Despite striking filamentation differences under laboratory conditions, however, analysis of cell morphology in the GI tract revealed that the three isolates exhibited similar filamentation properties in this in vivo niche. Notably, we found that SC5314 is more sensitive to the antimicrobial peptide CRAMP, and the use of CRAMP-deficient mice modestly increased the ability of SC5314 to colonize the GI tract relative to CHN1 and 529L. These studies provide new insights into how strain-specific differences impact C. albicans traits in the host and advance CHN1 and 529L as relevant strains to study C. albicans pathobiology in its natural host niche. IMPORTANCE Understanding how fungi colonize the GI tract is increasingly recognized as highly relevant to human health. The animal models used to study Candida albicans commensalism commonly rely on altering the host microbiome (via antibiotic treatment or defined diets) to establish successful GI colonization by the C. albicans reference isolate SC5314. Here, we characterize two C. albicans isolates that can colonize the murine GI tract without antibiotic treatment and can therefore be used as tools for studying fungal commensalism. Importantly, experiments were replicated in three different animal facilities and utilized three different mouse strains. Differential colonization between fungal isolates was not associated with alterations in the bacterial microbiome but rather with distinct responses to CRAMP, a host antimicrobial peptide. This work emphasizes the importance of C. albicans intraspecies variation as well as host antimicrobial defense mechanisms in defining the outcome of commensal interactions.

Comparative Proteomic Analysis of Histoplasma capsulatum Yeast and Mycelium Reveals Differential Metabolic Shifts and Cell Wall Remodeling Processes in the Different Morphotypes.

Histoplasma capsulatum is a thermally dimorphic fungus distributed worldwide, but with the highest incidence in the Americas within specific geographic areas, such as the Mississippi River Valley and regions in Latin America. This fungus is the etiologic agent of histoplasmosis, an important life-threatening systemic mycosis. Dimorphism is an important feature for fungal survival in different environments and is related to the virulence of H. capsulatum, and essential to the establishment of infection. Proteomic profiles have made important contributions to the knowledge of metabolism and pathogenicity in several biological models. However, H. capsulatum proteome studies have been underexplored. In the present study, we report the first proteomic comparison between the mycelium and the yeast cells of H. capsulatum. Liquid chromatography coupled to mass spectrometry was used to evaluate the proteomic profile of the two phases of H. capsulatum growth, mycelium, and yeast. In summary, 214 and 225 proteins were only detected/or preferentially abundant in mycelium or yeast cells, respectively. In mycelium, enzymes related to the glycolytic pathway and to the alcoholic fermentation occurred in greater abundance, suggesting a higher use of anaerobic pathways for energy production. In yeast cells, proteins related to the tricarboxylic acid cycle and response to temperature stress were in high abundance. Proteins related to oxidative stress response or involved with cell wall metabolism were identified with differential abundance in both conditions. Proteomic data validation was performed by enzymatic activity determination, Western blot assays, or immunofluorescence microscopy. These experiments corroborated, directly or indirectly, the abundance of isocitrate lyase, 2-methylcitrate synthase, catalase B, and mannosyl-oligosaccharide-1,2-alpha-mannosidase in the mycelium and heat shock protein (HSP) 30, HSP60, glucosamine-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, and N-acetylglucosamine-phosphate mutase in yeast cells. The proteomic profile-associated functional classification analyses of proteins provided new and interesting information regarding the differences in metabolism between the two distinct growth forms of H. capsulatum.

Large-scale transcriptomics to dissect 2 years of the life of a fungal phytopathogen interacting with its host plant.

BACKGROUND: The fungus Leptosphaeria maculans has an exceptionally long and complex relationship with its host plant, Brassica napus, during which it switches between different lifestyles, including asymptomatic, biotrophic, necrotrophic, and saprotrophic stages. The fungus is also exemplary of "two-speed" genome organisms in the genome of which gene-rich and repeat-rich regions alternate. Except for a few stages of plant infection under controlled conditions, nothing is known about the genes mobilized by the fungus throughout its life cycle, which may last several years in the field. RESULTS: We performed RNA-seq on samples corresponding to all stages of the interaction of L. maculans with its host plant, either alive or dead (stem residues after harvest) in controlled conditions or in field experiments under natural inoculum pressure, over periods of time ranging from a few days to months or years. A total of 102 biological samples corresponding to 37 sets of conditions were analyzed. We show here that about 9% of the genes of this fungus are highly expressed during its interactions with its host plant. These genes are distributed into eight well-defined expression clusters, corresponding to specific infection lifestyles or to tissue-specific genes. All expression clusters are enriched in effector genes, and one cluster is specific to the saprophytic lifestyle on plant residues. One cluster, including genes known to be involved in the first phase of asymptomatic fungal growth in leaves, is re-used at each asymptomatic growth stage, regardless of the type of organ infected. The expression of the genes of this cluster is repeatedly turned on and off during infection. Whatever their expression profile, the genes of these clusters are enriched in heterochromatin regions associated with H3K9me3 or H3K27me3 repressive marks. These findings provide support for the hypothesis that part of the fungal genes involved in niche adaptation is located in heterochromatic regions of the genome, conferring an extreme plasticity of expression. CONCLUSION: This work opens up new avenues for plant disease control, by identifying stage-specific effectors that could be used as targets for the identification of novel durable disease resistance genes, or for the in-depth analysis of chromatin remodeling during plant infection, which could be manipulated to interfere with the global expression of effector genes at crucial stages of plant infection.

A natural association of a yeast with Aspergillus terreus and its impact on the host fungal biology.

Several fungi have been shown to harbor microorganisms that regulate the key components of fungal metabolism. We explored the symbiotic association of an endophyte, Aspergillus terreus, which led to the isolation of a yeast, Meyerozyma caribbica, as its symbiont. An axenic fungal culture, free of the symbiont, was developed to study the effect of this association on the endophytic fungus. The symbiotic yeast partner was found to play an important role in the adaptation of A. terreus to thermal as well as osmotic stress. Under these stress conditions, the symbiont enhanced the production of lovastatin and the growth of the host fungus. The symbiotic yeast was found to induce the expression of the global regulator gene, the key genes involved in the lovastatin biosynthetic pathway as well as those involved in general growth and development, under stress conditions, in the fungal partner. Analysis by PCR and fluorescent in situ hybridization microscopy indicated that the yeast may be present inside the hyphae of the fungus. However, a direct method like transmission electron microscopy may help to better understand the dynamics of this association, including the distribution of the yeast cells in/on the fungal hyphae and spores.

Animal-associated fungi: Editorial.

Of 1882 fungal species described in 2019, only 3.5% were animal-associated. This percentage is representative of the poor understanding we have of this group of fungi, which are ephemeral, sometimes inconspicuous, and difficult to access, while often requiring specialized methods for their study. Following a two-session symposium on animal-associated fungi during the 2019 Annual Meeting of the Mycological Society of America, this special issue presents the work of 61 researchers in 16 countries. Twelve articles cover animal-associated fungi among Ascomycota, Basidiomycota, and Neocallimastigomycota-describing 29 new species, presenting new evolutionary hypotheses, and unearthing new ecological data.

Hülle Cells of Aspergillus nidulans with Nuclear Storage and Developmental Backup Functions Are Reminiscent of Multipotent Stem Cells.

Some aspergilli are among the most cosmopolitan and ecologically dominant fungal species. One pillar of their success is their complex life cycle, which creates specialized cell types for versatile dispersal and regenesis. One of these cell types is unique to aspergilli-the Hülle cells. Despite being known for over a century, the biological and ecological roles of Hülle cells remain largely speculative. Previously reported data on in vivo Hülle cell formation and localization have been conflicting. Our quantification reveals that Hülle cells can occur at all locations on hyphae and that they show cellular activity similar to that seen with adjacent hyphae, indicating that they develop as intricate parts of hyphal tissue. In addition, we show that during sexual development associated with two parental strains, the typically multinucleate Hülle cells can inherit nuclei from both parents, indicating that they may serve as genetic backups. We provide an easy, reproducible method to study Hülle cell biology and germination with which we investigate the 90-year-old puzzle of whether and how Hülle cells germinate. We present clear evidence for the germination of Hülle cells, and we show that Hülle cells grow hyphae that develop into a spore-producing colony. Finally, we show that Hülle cell-derived colonies produce conidiospores faster than spore-derived colonies, providing evidence for an as-yet-undescribed developmental shortcut program in Aspergillus nidulans We propose that Hülle cells represent a unique cell type as specialized hypha-derived sexual tissue with a nucleus storage function and may act as fungal backup stem cells under highly destructive conditions.IMPORTANCE The in vivo identification of Hülle cells in cases of aspergillosis infections in animals and humans illustrates their biological relevance and suggests that they might be involved in pathogenicity. It is striking that aspergilli have developed and maintained a multinucleate nurse cell that is presumably energy-intensive to produce and is usually found only in higher eukaryotes. Our findings shed light on how the understudied Hülle cells might contribute to the success of aspergilli by acting not only as nurse cells under detrimental conditions (sexual development) but also as fungal backup stem cells with the capacity to produce genetically diverse spores in an accelerated manner, thereby substantially contributing to survival in response to predator attack or under otherwise severely destructive conditions. Our study solved the 90-year-old puzzle of Hülle cell germination and provides easy, reproducible methods that will facilitate future studies on biological and ecological roles of Hülle cells in aspergilli.

Mating genes in Calonectria and evidence for a heterothallic ancestral state.

The genus Calonectria includes many important plant pathogens with a wide global distribution. In order to better understand the reproductive biology of these fungi, we characterised the structure of the mating type locus and flanking genes using the genome sequences for seven Calonectria species. Primers to amplify the mating type genes in other species were also developed. PCR amplification of the mating type genes and multi-gene phylogenetic analyses were used to investigate the mating strategies and evolution of mating type in a collection of 70 Calonectria species residing in 10 Calonectria species complexes. Results showed that the organisation of the MAT locus and flanking genes is conserved. In heterothallic species, a novel MAT gene, MAT1-2-12 was identified in the MAT1-2 idiomorph; the MAT1-1 idiomorph, in most cases, contained the MAT1-1-3 gene. Neither MAT1-1-3 nor MAT1-2-12 was found in homothallic Calonectria (Ca.) hongkongensis, Ca. lateralis, Ca. pseudoturangicola and Ca. turangicola. Four different homothallic MAT locus gene arrangements were observed. Ancestral state reconstruction analysis provided evidence that the homothallic state was basal in Calonectria and this evolved from a heterothallic ancestor.

Extracellular vesicles secreted by Saccharomyces cerevisiae are involved in cell wall remodelling.

Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1∆ or chs3∆ or the vps23∆chs3∆ double knockout strain were unable to rescue the yeast cells as compared to vps23∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.

Fungal acetylome comparative analysis identifies an essential role of acetylation in human fungal pathogen virulence.

Lysine acetylation is critical in regulating important biological processes in many organisms, yet little is known about acetylome evolution and its contribution to phenotypic diversity. Here, we compare the acetylomes of baker's yeast and the three deadliest human fungal pathogens, Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Using mass spectrometry enriched for acetylated peptides together with public data from Saccharomyces cerevisiae, we show that fungal acetylomes are characterized by dramatic evolutionary dynamics and limited conservation in core biological processes. Notably, the levels of protein acetylation in pathogenic fungi correlate with their pathogenicity. Using gene knockouts and pathogenicity assays in mice, we identify deacetylases with critical roles in virulence and protein translation elongation. Finally, through mutational analysis of deactylation motifs we find evidence of positive selection at specific acetylation motifs in fungal pathogens. These results shed new light on the pathogenicity regulation mechanisms underlying the evolution of fungal acetylomes.