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Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.

Gliotoxin (GT) plays a central role in the pathogenicity of Aspergillus fumigatus by enabling immune evasion and microbial competition, but its extreme toxicity also threatens the fungus itself. Although core GT biosynthetic and detoxification mechanisms are well studied, the full genetic network safeguarding against GT’s effects remains incompletely understood. This study identifies new RglT-regulated genes that contribute to GT self-protection and demonstrates that mitochondrial function is crucial for surviving GT exposure. Remarkably, similar protective pathways are active in both GT-producing and non-producing fungi, underscoring the ecological relevance of GT defense mechanisms. These findings deepen our understanding of fungal toxin tolerance and highlight mitochondria as a potential vulnerability that could be exploited for antifungal interventions.

Invasive fungal infections kill more than 1.5 million people each year, with limited treatment options. There is no vaccine available in clinical use to prevent and control fungal infections. Our recent studies showed that a mutant of the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase in Cryptococcus neoformans , elicited superior protective Th1 host immunity. Here, we demonstrate that the heat-killed fbp1 Δ cells (HK-fbp1) can be harnessed to confer protection against a challenge by the virulent parental strain, even in animals depleted of CD4 + T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that HK-fbp1 vaccination induces significant cross-protection against challenge with diverse invasive fungal pathogens. Thus, our data suggest that HK-fbp1 has the potential to be a broad-spectrum vaccine candidate against invasive fungal infections in both immunocompetent and immunocompromised populations. Cryptococcus neoformans is a fungal pathogen that infects the lungs and then often disseminates to the central nervous system, causing meningitis. How Cryptococcus is able to suppress host immunity and escape the antifungal activity of macrophages remains incompletely understood. We reported that the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase, promotes Cryptococcus virulence by regulating host- Cryptococcus interactions. Our recent studies demonstrated that the fbp1 Δ mutant elicited superior protective Th1 host immunity in the lungs and that the enhanced immunogenicity of heat-killed fbp1 Δ yeast cells can be harnessed to confer protection against a subsequent infection with the virulent parental strain. We therefore examined the use of heat-killed fbp1 Δ cells in several vaccination strategies. Interestingly, the vaccine protection remains effective even in mice depleted of CD4 + T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that vaccinating mice with heat-killed fbp1 Δ induces significant cross-protection against challenge with diverse invasive fungal pathogens, including C. neoformans , C. gattii , and Aspergillus fumigatus , as well as partial protection against Candida albicans . Thus, our data suggest that the heat-killed fbp1Δ strain has the potential to be a suitable vaccine candidate against cryptococcosis and other invasive fungal infections in both immunocompetent and immunocompromised populations. IMPORTANCE Invasive fungal infections kill more than 1.5 million people each year, with limited treatment options. There is no vaccine available in clinical use to prevent and control fungal infections. Our recent studies showed that a mutant of the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase in Cryptococcus neoformans , elicited superior protective Th1 host immunity. Here, we demonstrate that the heat-killed fbp1 Δ cells (HK-fbp1) can be harnessed to confer protection against a challenge by the virulent parental strain, even in animals depleted of CD4 + T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that HK-fbp1 vaccination induces significant cross-protection against challenge with diverse invasive fungal pathogens. Thus, our data suggest that HK-fbp1 has the potential to be a broad-spectrum vaccine candidate against invasive fungal infections in both immunocompetent and immunocompromised populations.

► NDV-3 is safe and generally well-tolerated in healthy adults. ► NDV-3 elicits quick and robust B- and T-cell immune responses. ► A single dose induces an anamnestic rather than priming immune response. ► T-cell response includes increases in PBMCs producing IFN-γ and/or IL-17A. ► A second dose further enhances the B- and T-cell immune response. The investigational vaccine, NDV-3, contains the N-terminal portion of the Candida albicans agglutinin-like sequence 3 protein (Als3p) formulated with an aluminum hydroxide adjuvant in phosphate-buffered saline. Preclinical studies demonstrated that the Als3p vaccine antigen protects mice from oropharyngeal, vaginal and intravenous challenge with C. albicans and other selected species of Candida as well as both intravenous challenge and skin and soft tissue infection with Staphylococcus aureus. The objectives of this first-in-human Phase I clinical trial were to evaluate the safety, tolerability and immunogenicity of NDV-3 at two different antigen levels compared to a saline placebo. Forty healthy, adult subjects were randomized to receive one dose of NDV-3 containing either 30 or 300μg of Als3p, or placebo. NDV-3 at both dose levels was safe and generally well-tolerated. Anti-Als3p total IgG and IgA1 levels for both doses reached peak levels by day 14 post vaccination, with 100% seroconversion of all vaccinated subjects. On average, NDV-3 stimulated peripheral blood mononuclear cell (PBMC) production of both IFN-γ and IL-17A, which peaked at day 7 for subjects receiving the 300μg dose and at day 28 for those receiving the 30μg dose. Six months after receiving the first dose of NDV-3, nineteen subjects received a second dose of NDV-3 identical to their first dose to evaluate memory B- and T-cell immune responses. The second dose resulted in a significant boost of IgG and IgA1 titers in >70% of subjects, with the biggest impact in those receiving the 30μg dose. A memory T-cell response was also noted for IFN-γ in almost all subjects and for IL-17A in the majority of subjects. These data support the continued investigation of NDV-3 as a vaccine candidate against Candida and S. aureus infections.

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.

Development of multicellular eukaryotes involves transcriptional reprogramming to drive cell fate transitions. This study identified PRC2 as a critical regulator of cell fate in the model filamentous fungus Neurospora crassa , where it silences a subset of sexual development genes. Loss of regulation by PRC2 triggers a major reprogramming event in which genes specifying sexual tissues cannot be repressed, causing a homeotic transition. These results provide novel insights into the role of PRC2-mediated regulation in the fungal kingdom and uncover a critical checkpoint regulating complex multicellular development.

Microbial effectors are small secreted proteins that help pathogens establish disease within the host environment. In biotrophic fungi, secreted chorismate mutases (CMs) like Cmu1 suppress the production of salicylic acid (SA), a key plant hormone involved in resistance against biotrophic pathogens. Since Cmu1 and its homologs are exclusively found in biotrophic pathogens, secreted CMs have been considered a hallmark of biotrophy. Surprisingly, we identified a secreted CM, encoded by SsCM1 , in the predominantly necrotrophic fungus Sclerotinia sclerotiorum . Structural and functional studies suggest SsCM1 is likely a functional homolog acquired from bacteria and specifically acts to suppress the production of antimicrobial compounds that would otherwise enhance plant resistance to necrotrophs. Unlike Cmu1, SsCM1 localizes to plastids, inversely regulates SA, and is conserved more broadly across the fungal kingdom. Thus, our findings reveal a new branch of plastid-localized CMs in necrotrophs, offering new avenues for the development of potential broad-spectrum antimicrobial treatments targeting this pathogen group.

A vaccine capable of protecting at-risk persons against infections due to Cryptococcus neoformans and Cryptococcus gattii could reduce the substantial global burden of human cryptococcosis. Vaccine development has been hampered though, by lack of knowledge as to which antigens are immunoprotective and the need for an effective vaccine delivery system. We made alkaline extracts from mutant cryptococcal strains that lacked capsule or chitosan. The extracts were then packaged into glucan particles (GPs), which are purified Saccharomyces cerevisiae cell walls composed primarily of β-1,3-glucans. Subcutaneous vaccination with the GP-based vaccines provided significant protection against subsequent pulmonary infection with highly virulent strains of C. neoformans and C. gattii . The alkaline extract derived from the acapsular strain was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), and the most abundant proteins were identified. Separation of the alkaline extract by size exclusion chromatography revealed fractions that conferred protection when loaded in GP-based vaccines. Robust Th1- and Th17-biased CD4 + T cell recall responses were observed in the lungs of vaccinated and infected mice. Thus, our preclinical studies have indicated promising cryptococcal vaccine candidates in alkaline extracts delivered in GPs. Ongoing studies are directed at identifying the individual components of the extracts that confer protection and thus would be promising candidates for a human vaccine. IMPORTANCE The encapsulated yeast Cryptococcus neoformans and its closely related sister species, Cryptococcus gattii , are major causes of morbidity and mortality, particularly in immunocompromised persons. This study reports on the preclinical development of vaccines to protect at-risk populations from cryptococcosis. Antigens were extracted from Cryptococcus by treatment with an alkaline solution. The extracted antigens were then packaged into glucan particles, which are hollow yeast cell walls composed mainly of β-glucans. The glucan particle-based vaccines elicited robust T cell immune responses and protected mice from otherwise-lethal challenge with virulent strains of C. neoformans and C. gattii . The technology used for antigen extraction and subsequent loading into the glucan particle delivery system is relatively simple and can be applied to vaccine development against other pathogens. The encapsulated yeast Cryptococcus neoformans and its closely related sister species, Cryptococcus gattii , are major causes of morbidity and mortality, particularly in immunocompromised persons. This study reports on the preclinical development of vaccines to protect at-risk populations from cryptococcosis. Antigens were extracted from Cryptococcus by treatment with an alkaline solution. The extracted antigens were then packaged into glucan particles, which are hollow yeast cell walls composed mainly of β-glucans. The glucan particle-based vaccines elicited robust T cell immune responses and protected mice from otherwise-lethal challenge with virulent strains of C. neoformans and C. gattii . The technology used for antigen extraction and subsequent loading into the glucan particle delivery system is relatively simple and can be applied to vaccine development against other pathogens.

Breathe easy: why inhaled fungal spores don't provoke an immune reaction Every day we inhale thousands of tiny fungal spores (conidia), originating from many different fungal species. Yet although these spores are packed with antigens and allergens, their inhalation does not continuously activate our innate immune cells or provoke inflammatory responses. A series of immunological, biochemical and genetic experiments shows why: immune recognition of these spores is prevented by a hydrophobic layer of rodlet proteins covering the conidial surface. If this layer is removed, spores activate the immune system. A pathogenic spore equipped with this defensive layer might lie dormant beyond host defences until conditions are suitable for germination. Therapeutically the robust nature of the rodlet proteins might be exploited to generate nanoparticles containing embedded molecules targeted to a specific location in the body, or optimized for sustained delivery. Fungal spores are ubiquitous in the air we breathe and contain many antigens and allergens, and yet they neither continuously activate the host innate immune cells nor induce detrimental inflammatory responses after their inhalation. Here, the surface layer on dormant spores is shown to mask their recognition by the immune system and hence prevent an immune response. The air we breathe is filled with thousands of fungal spores (conidia) per cubic metre, which in certain composting environments can easily exceed 10 9 per cubic metre. They originate from more than a hundred fungal species belonging mainly to the genera Cladosporium , Penicillium , Alternaria and Aspergillus 1 , 2 , 3 , 4 . Although these conidia contain many antigens and allergens 5 , 6 , 7 , it is not known why airborne fungal microflora do not activate the host innate immune cells continuously and do not induce detrimental inflammatory responses following their inhalation. Here we show that the surface layer on the dormant conidia masks their recognition by the immune system and hence prevents immune response. To explore this, we used several fungal members of the airborne microflora, including the human opportunistic fungal pathogen Aspergillus fumigatus , in in vitro assays with dendritic cells and alveolar macrophages and in in vivo murine experiments. In A. fumigatus , this surface ‘rodlet layer’ is composed of hydrophobic RodA protein covalently bound to the conidial cell wall through glycosylphosphatidylinositol-remnants. RodA extracted from conidia of A. fumigatus was immunologically inert and did not induce dendritic cell or alveolar macrophage maturation and activation, and failed to activate helper T-cell immune responses in vivo . The removal of this surface ‘rodlet/hydrophobin layer’ either chemically (using hydrofluoric acid), genetically (Δ rodA mutant) or biologically (germination) resulted in conidial morphotypes inducing immune activation. All these observations show that the hydrophobic rodlet layer on the conidial cell surface immunologically silences airborne moulds.

The ribosomal RNA (rDNA) gene repeats are essential housekeeping genes found in all organisms. A gene amplification system maintains large cluster(s) of tandemly repeated copies in the chromosome, with each species having a specific number of copies. Yeast has many untranscribed rDNA copies (extra copies), and we found that when they are lost, the cells become sensitive to DNA damage induced by mutagens. We show that this sensitivity is dependent on rDNA transcriptional activity, which interferes with cohesion between rDNA loci of sister chromatids. The extra rDNA copies facilitate condensin association and sister-chromatid cohesion, thereby facilitating recombinational repair. These results suggest that high concentrations of heavily transcribed genes are toxic to the cells, and therefore amplified genes, such as rDNA, have evolved.