Journal of Fungi

19 pages, 10196 KiB

Open AccessEditor’s ChoiceArticle

A Chimeric ORF Fusion Phenotypic Reporter for Cryptococcus neoformans

by Louis S. Phillips-Rose, Chendi K. Yu, Nicholas P. West and James A. Fraser

J. Fungi 2024, 10(8), 567; https://doi.org/10.3390/jof10080567 - 12 Aug 2024

Viewed by 1656

The plethora of genome sequences produced in the postgenomic age has not resolved many of our most pressing biological questions. Correlating gene expression with an interrogatable and easily observable characteristic such as the surrogate phenotype conferred by a reporter gene is a valuable [...] Read more.

The plethora of genome sequences produced in the postgenomic age has not resolved many of our most pressing biological questions. Correlating gene expression with an interrogatable and easily observable characteristic such as the surrogate phenotype conferred by a reporter gene is a valuable approach to gaining insight into gene function. Many reporters including lacZ, amdS, and the fluorescent proteins mRuby3 and mNeonGreen have been used across all manners of organisms. Described here is an investigation into the creation of a robust, synthetic, fusion reporter system for Cryptococcus neoformans that combines some of the most useful fluorophores available in this system with the versatility of the counter-selectable nature of amdS. The reporters generated include multiple composition and orientation variants, all of which were investigated for differences in expression. Evaluation of known promoters from the TEF1 and GAL7 genes was undertaken, elucidating novel expression tendencies of these biologically relevant C. neoformans regulators of transcription. Smaller than lacZ but providing multiple useful surrogate phenotypes for interrogation, the fusion ORF serves as a superior whole-cell assay compared to traditional systems. Ultimately, the work described here bolsters the array of relevant genetic tools that may be employed in furthering manipulation and understanding of the WHO fungal priority group pathogen C. neoformans. Full article

(This article belongs to the Special Issue Molecular Processes of Fungi, 2nd Edition)

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14 pages, 5662 KiB

Open AccessEditor’s ChoiceArticle

The Mechanosensitive Pkd2 Channel Modulates the Recruitment of Myosin II and Actin to the Cytokinetic Contractile Ring

by Pritha Chowdhury, Debatrayee Sinha, Abhishek Poddar, Madhurya Chetluru and Qian Chen

J. Fungi 2024, 10(7), 455; https://doi.org/10.3390/jof10070455 - 28 Jun 2024

Cited by 2 | Viewed by 1394

Abstract

Cytokinesis, the last step in cell division, separates daughter cells through mechanical force. This is often through the force produced by an actomyosin contractile ring. In fission yeast cells, the ring helps recruit a mechanosensitive ion channel, Pkd2, to the cleavage furrow, whose [...] Read more.

Cytokinesis, the last step in cell division, separates daughter cells through mechanical force. This is often through the force produced by an actomyosin contractile ring. In fission yeast cells, the ring helps recruit a mechanosensitive ion channel, Pkd2, to the cleavage furrow, whose activation by membrane tension promotes calcium influx and daughter cell separation. However, it is unclear how the activities of Pkd2 may affect the actomyosin ring. Here, through both microscopic and genetic analyses of a hypomorphic pkd2 mutant, we examined the potential role of this essential gene in assembling the contractile ring. The pkd2-81KD mutation significantly increased the counts of the type II myosin heavy chain Myo2 (+18%), its regulatory light chain Rlc1 (+37%) and actin (+100%) molecules in the ring, compared to the wild type. Consistent with a regulatory role of Pkd2 in the ring assembly, we identified a strong negative genetic interaction between pkd2-81KD and the temperature-sensitive mutant myo2-E1. The pkd2-81KD myo2-E1 cells often failed to assemble a complete contractile ring. We conclude that Pkd2 modulates the recruitment of type II myosin and actin to the contractile ring, suggesting a novel calcium-dependent mechanism regulating the actin cytoskeletal structures during cytokinesis. Full article

(This article belongs to the Special Issue Yeast Cytokinesis)

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12 pages, 8293 KiB

Open AccessEditor’s ChoiceArticle

The Near-Gapless Penicillium fuscoglaucum Genome Enables the Discovery of Lifestyle Features as an Emerging Post-Harvest Phytopathogen

by Dianiris Luciano-Rosario, Wayne M. Jurick II and Christopher Gottschalk

J. Fungi 2024, 10(6), 430; https://doi.org/10.3390/jof10060430 - 18 Jun 2024

Cited by 1 | Viewed by 1658

Abstract

Penicillium spp. occupy many diverse biological niches that include plant pathogens, opportunistic human pathogens, saprophytes, indoor air contaminants, and those selected specifically for industrial applications to produce secondary metabolites and lifesaving antibiotics. Recent phylogenetic studies have established Penicillium fuscoglaucum as a synonym for [...] Read more.

Penicillium spp. occupy many diverse biological niches that include plant pathogens, opportunistic human pathogens, saprophytes, indoor air contaminants, and those selected specifically for industrial applications to produce secondary metabolites and lifesaving antibiotics. Recent phylogenetic studies have established Penicillium fuscoglaucum as a synonym for Penicillium commune, which is an indoor air contaminant and toxin producer and can infect apple fruit during storage. During routine culturing on selective media in the lab, we obtained an isolate of P. fuscoglaucum Pf_T2 and sequenced its genome. The Pf_T2 genome is far superior to available genomic resources for the species. Our assembly exhibits a length of 35.1 Mb, a BUSCO score of 97.9% complete, and consists of five scaffolds/contigs representing the four expected chromosomes. It was determined that the Pf_T2 genome was colinear with a type specimen P. fuscoglaucum and contained a lineage-specific, intact cyclopiazonic acid (CPA) gene cluster. For comparison, a highly virulent postharvest apple pathogen, P. expansum strain TDL 12.1, was included and showed a similar growth pattern in culture to our Pf_T2 isolate but was far more aggressive in apple fruit than P. fuscoglaucum. The genome of Pf_T2 serves as a major improvement over existing resources, has superior annotation, and can inform forthcoming omics-based work and functional genetic studies to probe secondary metabolite production and disparities in aggressiveness during apple fruit decay. Full article

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29 pages, 27808 KiB

Open AccessEditor’s ChoiceArticle

Sunken Riches: Ascomycete Diversity in the Western Mediterranean Coast through Direct Plating and Flocculation, and Description of Four New Taxa

by Daniel Guerra-Mateo, José F. Cano-Lira, Ana Fernández-Bravo and Josepa Gené

J. Fungi 2024, 10(4), 281; https://doi.org/10.3390/jof10040281 - 11 Apr 2024

Cited by 1 | Viewed by 2236

Abstract

The Mediterranean Sea stands out as a hotspot of biodiversity, whose fungal composition remains underexplored. Marine sediments represent the most diverse substrate; however, the challenge of recovering fungi in culture hinders the precise identification of this diversity. Concentration techniques like skimmed milk flocculation [...] Read more.

The Mediterranean Sea stands out as a hotspot of biodiversity, whose fungal composition remains underexplored. Marine sediments represent the most diverse substrate; however, the challenge of recovering fungi in culture hinders the precise identification of this diversity. Concentration techniques like skimmed milk flocculation (SMF) could represent a suitable solution. Here, we compare the effectiveness in recovering filamentous ascomycetes of direct plating and SMF in combination with three culture media and two incubation temperatures, and we describe the fungal diversity detected in marine sediments. Sediments were collected at different depths on two beaches (Miracle and Arrabassada) on the Spanish western Mediterranean coast between 2021 and 2022. We recovered 362 strains, and after a morphological selection, 188 were identified primarily with the LSU and ITS barcodes, representing 54 genera and 94 species. Aspergillus, Penicillium, and Scedosporium were the most common genera, with different percentages of abundance between both beaches. Arrabassada Beach was more heterogeneous, with 42 genera representing 60 species (Miracle Beach, 28 genera and 54 species). Although most species were recovered with direct plating (70 species), 20 species were exclusively obtained using SMF as a sample pre-treatment, improving our ability to detect fungi in culture. In addition, we propose three new species in the genera Exophiala, Nigrocephalum, and Queenslandipenidiella, and a fourth representing the novel genus Schizochlamydosporiella. We concluded that SMF is a useful technique that, in combination with direct plating, including different culture media and incubation temperatures, improves the chance of recovering marine fungal communities in culture-dependent studies. Full article

(This article belongs to the Special Issue Diversity and Ecology of Fungi from Underexplored and Extreme Environments)

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22 pages, 5491 KiB

Open AccessEditor’s ChoiceArticle

Characterization of Microbotryum lychnidis-dioicae Secreted Effector Proteins, Their Potential Host Targets, and Localization in a Heterologous Host Plant

by Ming-Chang Tsai, Michelle T. Barati, Venkata S. Kuppireddy, William C. Beckerson, Grace Long and Michael H. Perlin

J. Fungi 2024, 10(4), 262; https://doi.org/10.3390/jof10040262 - 30 Mar 2024

Cited by 1 | Viewed by 1723

Abstract

Microbotryum lychnidis-dioicae is an obligate fungal species colonizing the plant host, Silene latifolia. The fungus synthesizes and secretes effector proteins into the plant host during infection to manipulate the host for completion of the fungal lifecycle. The goal of this study was [...] Read more.

Microbotryum lychnidis-dioicae is an obligate fungal species colonizing the plant host, Silene latifolia. The fungus synthesizes and secretes effector proteins into the plant host during infection to manipulate the host for completion of the fungal lifecycle. The goal of this study was to continue functional characterization of such M. lychnidis-dioicae effectors. Here, we identified three putative effectors and their putative host-plant target proteins. MVLG_02245 is highly upregulated in M. lychnidis-dioicae during infection; yeast two-hybrid analysis suggests it targets a tubulin α-1 chain protein ortholog in the host, Silene latifolia. A potential plant protein interacting with MVLG_06175 was identified as CASP-like protein 2C1 (CASPL2C1), which facilitates the polymerization of the Casparian strip at the endodermal cells. Proteins interacting with MVLG_05122 were identified as CSN5a or 5b, involved in protein turnover. Fluorescently labelled MVLG_06175 and MVLG_05122 were expressed in the heterologous plant, Arabidopsis thaliana. MVLG_06175 formed clustered granules at the tips of trichomes on leaves and in root caps, while MVLG_05122 formed a band structure at the base of leaf trichomes. Plants expressing MVLG_05122 alone were more resistant to infection with Fusarium oxysporum. These results indicate that the fungus might affect the formation of the Casparian strip in the roots and the development of trichomes during infection as well as alter plant innate immunity. Full article

(This article belongs to the Special Issue Functional Understanding of Smut Biology)

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13 pages, 3080 KiB

Open AccessEditor’s ChoiceArticle

Clinical Diagnosis and Laboratory Testing of Abnormal Appearing Toenails: A Retrospective Assessment of Confirmatory Testing for Onychomycosis in the United States, 2022–2023

by Aditya K. Gupta, Tong Wang, Elizabeth A. Cooper, Sara A. Lincoln, Hui-Chen Foreman, William P. Scherer and Wayne L. Bakotic

J. Fungi 2024, 10(2), 149; https://doi.org/10.3390/jof10020149 - 13 Feb 2024

Cited by 10 | Viewed by 3499

Abstract

Onychomycosis is an under-recognized healthcare burden. Despite the risk of misdiagnosis, confirmatory laboratory testing is under-utilized. Histopathologic examination with polymerase chain reaction (PCR) is currently the most effective diagnostic method; it offers direct detection and identification of a fungal invasion. In this retrospective [...] Read more.

Onychomycosis is an under-recognized healthcare burden. Despite the risk of misdiagnosis, confirmatory laboratory testing is under-utilized. Histopathologic examination with polymerase chain reaction (PCR) is currently the most effective diagnostic method; it offers direct detection and identification of a fungal invasion. In this retrospective cohort study, we assessed confirmatory testing results, with matching clinical diagnoses, in 96,293 nail specimens submitted during a 9-month period from 2022 to 2023. Toenail specimens were examined using fungal culture, histopathology and/or PCR. Clinical diagnoses were identified using the International Classification of Diseases 10th Revision codes. For clinically diagnosed onychomycosis patients, the overall positivity rate was 59.4%; a similar positivity rate (59.5%) was found in patients with clinically diagnosed non-fungal nail dystrophy. Performing a histopathologic examination with PCR was more likely to provide pathogen identification results than using fungal culture. Male patients had a higher rate of onychomycosis overall; however, female patients had more non-dermatophyte mold onychomycosis caused by Aspergillus. Clinically diagnosed onychomycosis patients with a co-diagnosis of tinea pedis were more likely to test positive for onychomycosis by PCR (odds ratio [OR]: 4.2; 95% confidence interval [CI]: 2.7–6.4), histopathology (OR: 2.5; 95% CI: 2.0–3.1) and fungal culture (OR: 3.2; 95% CI: 1.5–6.6). Our results support the use of confirmatory laboratory testing when there is a clinical diagnosis of onychomycosis. Full article

(This article belongs to the Special Issue New Perspectives for Superficial Fungal Infections, Second Edition)

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16 pages, 2580 KiB

Open AccessEditor’s ChoiceArticle

Snake Fungal Disease in Free-Ranging Northern Pine Snakes (Pituophis melanoleucus melanoleucus) in New Jersey: Lesions, Severity of Sores and Investigator’s Perceptions

by Joanna Burger, Christian Jeitner, Robert T. Zappalorti, John F. Bunnell, Kelly Ng, Emile DeVito, David Schneider and Michael Gochfeld

J. Fungi 2024, 10(2), 125; https://doi.org/10.3390/jof10020125 - 3 Feb 2024

Cited by 2 | Viewed by 2023

Abstract

Ophidiomyces ophidiicola, the fungus causing snake fungal disease (SFD), has been identified in northern pine snakes (Pituophis melanoleucus) in New Jersey. In this paper, we (1) review the positivity rate of SFD on different locations on snakes’ bodies, (2) determine [...] Read more.

Ophidiomyces ophidiicola, the fungus causing snake fungal disease (SFD), has been identified in northern pine snakes (Pituophis melanoleucus) in New Jersey. In this paper, we (1) review the positivity rate of SFD on different locations on snakes’ bodies, (2) determine the relationship between the sores and quantitative polymerase chain reaction (qPCR) positivity rates, and (3) explore the relationship between the investigators’ clinical evaluation of the severity of sores, their evaluation of the likelihood of the sores being positive, and the qPCR positivity of SFD for the sores. Swabbing the sores was more effective at determining whether the snakes tested positive for O. ophidiicola than ventrum swabbing alone. The perception of the severity of the sores did not relate to qPCR positivity for O. ophidiicola. We suggest that the assessment of the rate of SFD among snakes in the wild needs to include the sampling of snakes with no clinical signs, as well as those with sores, and the swabbing of all the sores collectively. Clear terminology for sores, the identification of clinical signs of SFD, and distinguishing the rates of O. ophidiicola by PCR testing should be adopted. Overall, the pine snakes exhibited a higher rate of sores and positivity of O. ophidiicola swabs by PCR testing compared to the other snakes. Full article

(This article belongs to the Special Issue Fungal Diseases in Animals, 2nd Edition)

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28 pages, 2379 KiB

Open AccessEditor’s ChoiceArticle

Fungal Endophytes: Discovering What Lies within Some of Canada’s Oldest and Most Resilient Grapevines

by Shawkat Ali, A. Harrison Wright, Joey B. Tanney, Justin B. Renaud and Mark W. Sumarah

J. Fungi 2024, 10(2), 105; https://doi.org/10.3390/jof10020105 - 26 Jan 2024

Viewed by 2325

Abstract

Plant diseases and pests reduce crop yields, accounting for global crop losses of 30% to 50%. In conventional agricultural production systems, these losses are typically controlled by applying chemical pesticides. However, public pressure is mounting to curtail agrochemical use. In this context, employing [...] Read more.

Plant diseases and pests reduce crop yields, accounting for global crop losses of 30% to 50%. In conventional agricultural production systems, these losses are typically controlled by applying chemical pesticides. However, public pressure is mounting to curtail agrochemical use. In this context, employing beneficial endophytic microorganisms is an increasingly attractive alternative to the use of conventional chemical pesticides in agriculture. A multitude of fungal endophytes are naturally present in plants, producing enzymes, small peptides, and secondary metabolites due to their bioactivity, which can protect hosts from pathogens, pests, and abiotic stresses. The use of beneficial endophytic microorganisms in agriculture is an increasingly attractive alternative to conventional pesticides. The aim of this study was to characterize fungal endophytes isolated from apparently healthy, feral wine grapes in eastern Canada that have grown without agrochemical inputs for decades. Host plants ranged from unknown seedlings to long-lost cultivars not widely propagated since the 1800s. HPLC-MS was used to identify unique endophyte-derived chemical compounds in the host plants, while dual-culture competition assays showed a range in endophytes’ ability to suppress the mycelial growth of Botrytis, which is typically controlled in viticulture with pesticides. Twelve of the most promising fungal endophytes isolated were identified using multilocus sequencing and morphology, while DNA barcoding was employed to identify some of their host vines. These fungal endophyte isolates, which consisted of both known and putative novel strains, belonged to seven genera in six families and five orders of Ascomycota. Exploring the fungal endophytes in these specimens may yield clues to the vines’ survival and lead to the discovery of novel biocontrol agents. Full article

(This article belongs to the Special Issue Fungal Endophytes in Agriculture)

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14 pages, 43350 KiB

Open AccessEditor’s ChoiceArticle

Genetic Analysis of Candida albicans Filamentation by the Iron Chelator BPS Reveals a Role for a Conserved Kinase—WD40 Protein Pair

by Mariel Pinsky and Daniel Kornitzer

J. Fungi 2024, 10(1), 83; https://doi.org/10.3390/jof10010083 - 22 Jan 2024

Cited by 1 | Viewed by 2104

Abstract

Candida albicans is a major human pathogenic fungus that is distinguished by its capability to switch from a yeast to a hyphal morphology under different conditions. Here, we analyze the cellular effects of high concentrations of the iron chelator bathophenanthroline disulfonate (BPS). BPS [...] Read more.

Candida albicans is a major human pathogenic fungus that is distinguished by its capability to switch from a yeast to a hyphal morphology under different conditions. Here, we analyze the cellular effects of high concentrations of the iron chelator bathophenanthroline disulfonate (BPS). BPS inhibits cellular growth by withholding iron, but when iron chelation is overcome by the addition of hemoglobin as an iron source, the cells resume growth as hyphae. The BPS hyphal induction pathway was characterized by identifying the hyphal-specific transcription factors that it requires and by a forward genetic screen for mutants that fail to form hyphae in BPS using a transposon library generated in a haploid strain. Among the mutants identified are the DYRK1-like kinase Yak1 and Orf19.384, a homolog of the DYRK1-associated protein WDR68/DCAF7. Orf19.384 nuclear localization depends on Yak1, similar to their mammalian counterparts. We identified the hyphal suppressor transcription factor Sfl1 as a candidate target of Yak1-Orf19.384 and show that Sfl1 modification is similarly affected in the yak1 and orf19.384 mutant strains. These results suggest that DYRK1/Yak1 and WDR68/Orf19.384 represent a conserved protein pair that regulates cell differentiation from fungi to animals. Full article

(This article belongs to the Section Fungal Genomics, Genetics and Molecular Biology)

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20 pages, 4910 KiB

Open AccessEditor’s ChoiceArticle

Specific Phylotypes of Saprolegnia parasitica Associated with Atlantic Salmon Freshwater Aquaculture

by Kypher Varin Shreves, Marcia Saraiva, Tahmina Ruba, Claire Miller, E. Marian Scott, Debbie McLaggan and Pieter van West

J. Fungi 2024, 10(1), 57; https://doi.org/10.3390/jof10010057 - 9 Jan 2024

Cited by 2 | Viewed by 2553

Abstract

Saprolegniosis is a major destructive disease in freshwater aquaculture. The destructive economic impact of saprolegniosis on freshwater aquaculture necessitates further study on the range of Saprolegnia species within Atlantic salmon fish farms. This study undertook a thorough analysis of a total of 412 [...] Read more.

Saprolegniosis is a major destructive disease in freshwater aquaculture. The destructive economic impact of saprolegniosis on freshwater aquaculture necessitates further study on the range of Saprolegnia species within Atlantic salmon fish farms. This study undertook a thorough analysis of a total of 412 oomycete and fungal isolates that were successfully cultured and sequenced from 14 aquaculture sites in Scotland across a two-year sampling period. An ITS phylogenetic analysis of all isolates was performed according to whether they were isolated from fish or water samples and during enzootic or epizootic periods. Several genera of oomycetes were isolated from sampling sites, including Achlya, Leptolegnia, Phytophthora, and Pythium, but by far the most prevalent was Saprolegnia, accounting for 66% of all oomycetes isolated. An analysis of the ITS region of Saprolegnia parasitica showed five distinct phylotypes (S2–S6); S1 was not isolated from any site. Phylotype S2 was the most common and most widely distributed phylotype, being found at 12 of the 14 sampling sites. S2 was overwhelmingly sampled from fish (93.5%) and made up 91.1% of all S. parasitica phylotypes sampled during epizootics, as well as 67.2% of all Saprolegnia. This study indicates that a single phylotype may be responsible for Saprolegnia outbreaks in Atlantic salmon fish farms, and that water sampling and spore counts alone may be insufficient to predict Saprolegnia outbreaks in freshwater aquaculture. Full article

(This article belongs to the Special Issue Fungal Infections in Fishes and Aquatic Invertebrates, Second Edition)

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11 pages, 2138 KiB

Open AccessEditor’s ChoiceArticle

Phase-Dependent Differential In Vitro and Ex Vivo Susceptibility of Aspergillus flavus and Fusarium keratoplasticum to Azole Antifungals

by Darby Roberts, Jacklyn Salmon, Marc A. Cubeta and Brian C. Gilger

J. Fungi 2023, 9(10), 966; https://doi.org/10.3390/jof9100966 - 26 Sep 2023

Cited by 2 | Viewed by 1499

Abstract

Fungal keratitis (FK) is an invasive infection of the cornea primarily associated with Aspergillus and Fusarium species. FK is treated empirically with a limited selection of topical antifungals with varying levels of success. Though clinical infections are typically characterized by a dense network [...] Read more.

Fungal keratitis (FK) is an invasive infection of the cornea primarily associated with Aspergillus and Fusarium species. FK is treated empirically with a limited selection of topical antifungals with varying levels of success. Though clinical infections are typically characterized by a dense network of mature mycelium, traditional models used to test antifungal susceptibility of FK isolates exclusively evaluate susceptibility in fungal cultures derived from asexual spores known as conidia. The purpose of this study was to characterize differences in fungal response when topical antifungal treatment is initiated at progressive phases of fungal development. We compared the efficacy of voriconazole and luliconazole against in vitro cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. A porcine cadaver corneal model was used to compare antifungal efficacy of voriconazole and luliconazole in ex vivo tissue cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. Our results demonstrate phase-dependent susceptibility of both A. flavus and F. keratoplasticum to both azoles in vitro as well as ex vivo. We conclude that traditional antifungal susceptibility testing with conidial suspensions does not correlate with fungal susceptibility in cultures of a more advanced developmental phase. A revised method of antifungal susceptibility testing that evaluates hyphal susceptibility may better predict fungal response in the clinical setting where treatment is often delayed until days after the initial insult. Full article

(This article belongs to the Special Issue Fungal Diseases in Animals, 2nd Edition)

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26 pages, 2245 KiB

Open AccessEditor’s ChoiceArticle

Spectrometric Characterization of Clinical and Environmental Isolates of Aspergillus Series Versicolores

by Océane Jomat, Antoine Géry, Astrid Leudet, Agathe Capitaine, David Garon and Julie Bonhomme

J. Fungi 2023, 9(9), 868; https://doi.org/10.3390/jof9090868 - 23 Aug 2023

Viewed by 1792

Abstract

Aspergillus series Versicolores are molds distributed among 17 species, commonly found in our environment, and responsible for infections. Since 2022, a new taxonomy has grouped them into 4 major lineages: A. versicolor, A. subversicolor, A. sydowii, and A. creber. [...] Read more.

Aspergillus series Versicolores are molds distributed among 17 species, commonly found in our environment, and responsible for infections. Since 2022, a new taxonomy has grouped them into 4 major lineages: A. versicolor, A. subversicolor, A. sydowii, and A. creber. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) could be a faster and more cost-effective alternative to molecular techniques for identifying them by developing a local database. To evaluate this technique, 30 isolates from Aspergillus series Versicolores were used. A total of 59 main spectra profiles (MSPs) were created in the local database. This protocol enabled accurate identification of 100% of the extracted isolates, of which 97% (29/30) were correctly identified with a log score ≥ 2.00. Some MSPs recorded as Aspergillus versicolor in the supplier’s database could lead to false identifications as they did not match with the correct lineages. Although the local database is still limited in the number and diversity of species of Aspergillus series Versicolores, it is sufficiently effective for correct lineage identification according to the latest taxonomic revision, and better than the MALDI-TOF MS supplier’s database. This technology could improve the speed and accuracy of routine fungal identification for these species. Full article

(This article belongs to the Special Issue Recent Advances in Taxonomy, Phylogeny and Evolution of Fungi)

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15 pages, 3704 KiB

Open AccessEditor’s ChoiceArticle

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Plant Biomass-Degrading Strategies in Fungi

by Jiajia Li, Ad Wiebenga, Anna Lipzen, Vivian Ng, Sravanthi Tejomurthula, Yu Zhang, Igor V. Grigoriev, Mao Peng and Ronald P. de Vries

J. Fungi 2023, 9(8), 860; https://doi.org/10.3390/jof9080860 - 18 Aug 2023

Cited by 9 | Viewed by 3324

Abstract

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as [...] Read more.

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as cellulases, hemicellulases, and pectinases. Due to the crucial role of plant biomass-degrading (PBD) CAZymes in fungal growth and related biotechnology applications, investigation of their genomic diversity and transcriptional dynamics has attracted increasing attention. In this project, we systematically compared the genome content of PBD CAZymes in six taxonomically distant species, Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium, and Dichomitus squalens, as well as their transcriptome profiles during growth on nine monosaccharides. Considerable genomic variation and remarkable transcriptomic diversity of CAZymes were identified, implying the preferred carbon source of these fungi and their different methods of transcription regulation. In addition, the specific carbon utilization ability inferred from genomics and transcriptomics was compared with fungal growth profiles on corresponding sugars, to improve our understanding of the conversion process. This study enhances our understanding of genomic and transcriptomic diversity of fungal plant polysaccharide-degrading enzymes and provides new insights into designing enzyme mixtures and metabolic engineering of fungi for related industrial applications. Full article

(This article belongs to the Special Issue Filamentous Fungi as Excellent Industrial Strains: Development and Applications)

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20 pages, 3014 KiB

Open AccessEditor’s ChoiceArticle

The Potential of Wild Yeasts as Promising Biocontrol Agents against Pine Canker Diseases

by Eugenia Iturritxa, Nebai Mesanza and María-Jesús Torija

J. Fungi 2023, 9(8), 840; https://doi.org/10.3390/jof9080840 - 11 Aug 2023

Cited by 2 | Viewed by 1781

Abstract

Native wild yeasts from forest ecosystems represent an interesting potential source of biocontrol organisms in synergy with disease-tolerant forest materials. Yeasts have a combination of competitive mechanisms and low requirements for their biotechnological application as biocontrol agents. The current study aimed to increase [...] Read more.

Native wild yeasts from forest ecosystems represent an interesting potential source of biocontrol organisms in synergy with disease-tolerant forest materials. Yeasts have a combination of competitive mechanisms and low requirements for their biotechnological application as biocontrol agents. The current study aimed to increase the number of biocontrol candidates against Fusarium circinatum and Diplodia sapinea. The enzymatic and antagonistic activities of the biocontrol candidates were evaluated using different screening methods, in which the direct impact on the growth of the pathogen was measured as well as some properties such as cellulose and lignin degradation, tolerance to biocides, volatile compound production, or iron effect, which may be of interest in biotechnological processes related to the management of forest diseases. A total of 58 yeast strains belonging to 21 different species were obtained from oak forest and vineyard ecosystems and evaluated. The application of yeast treatment behaved differently depending on the pathogen and the plant clone. The 2g isolate (Torulaspora delbrueckii) showed the highest inhibitory activity for D. sapinea and 25q and 90q (Saccharomyces paradoxus) for F. circinatum. Clones IN416 and IN216 were the most susceptible and the most tolerant to D. sapinea, respectively, while the opposite was observed for F. circinatum. Full article

(This article belongs to the Special Issue Fungal Pathogens and Host Plants)

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20 pages, 3892 KiB

Open AccessEditor’s ChoiceArticle

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones

by Carmen Hicks, Thomas E. Witte, Amanda Sproule, Anne Hermans, Samuel W. Shields, Ronan Colquhoun, Chris Blackman, Christopher N. Boddy, Rajagopal Subramaniam and David P. Overy

J. Fungi 2023, 9(7), 695; https://doi.org/10.3390/jof9070695 - 23 Jun 2023

Cited by 5 | Viewed by 2751

Abstract

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their [...] Read more.

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as “C16” are likely associated with pathogenicity. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS² experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors. Full article

(This article belongs to the Special Issue Genome Editing Tools in Fungi)

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20 pages, 1548 KiB

Open AccessEditor’s ChoiceArticle

Clorgyline Analogs Synergize with Azoles against Drug Efflux in Candida auris

by Stephanie Toepfer, Michaela Lackner, Mikhail V. Keniya, Lisa-Maria Zenz, Marianne Friemert, Franz Bracher and Brian C. Monk

J. Fungi 2023, 9(6), 663; https://doi.org/10.3390/jof9060663 - 13 Jun 2023

Cited by 4 | Viewed by 2996

Abstract

Concern about the global emergence of multidrug-resistant fungal pathogens led us to explore the use of combination therapy to combat azole resistance in Candida auris. Clorgyline had previously been shown to be a multi-target inhibitor of Cdr1 and Mdr1 efflux pumps of [...] Read more.

Concern about the global emergence of multidrug-resistant fungal pathogens led us to explore the use of combination therapy to combat azole resistance in Candida auris. Clorgyline had previously been shown to be a multi-target inhibitor of Cdr1 and Mdr1 efflux pumps of Candida albicans and Candida glabrata. A screen for antifungal sensitizers among synthetic analogs of Clorgyline detected interactions with the C. auris efflux pump azole substrates Posaconazole and Voriconazole. Of six Clorgyline analogs, M19 and M25 were identified as potential sensitizers of azole resistance. M19 and M25 were found to act synergistically with azoles against resistant C. auris clade I isolates and recombinant Saccharomyces cerevisiae strains overexpressing C. auris efflux pumps. Nile Red assays with the recombinant strains showed M19 and M25 inhibited the activity of Cdr1 and Mdr1 efflux pumps that are known to play key roles in azole resistance in C. auris clades I, III, and IV. While Clorgyline, M19 and M25 uncoupled the Oligomycin-sensitive ATPase activity of Cdr1 from C. albicans and C. auris, their mode of action is yet to be fully elucidated. The experimental combinations described herein provides a starting point to combat azole resistance dominated by overexpression of CauCdr1 in C. auris clades I and IV and CauMdr1 in C. auris clade III. Full article

(This article belongs to the Special Issue Antifungal Drug Discovery: Novel Therapies and Approaches)

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17 pages, 1460 KiB

Open AccessEditor’s ChoiceArticle

Mutations in the Second Alternative Oxidase Gene: A New Approach to Group Aspergillus niger Strains

by Michel Flipphi, Alexandra Márton, Vivien Bíró, Norbert Ág, Erzsébet Sándor, Erzsébet Fekete and Levente Karaffa

J. Fungi 2023, 9(5), 570; https://doi.org/10.3390/jof9050570 - 13 May 2023

Cited by 1 | Viewed by 2256

Abstract

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two [...] Read more.

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two divergent species of the subgenus Nidulantes—A. calidoustus and A. implicatus—as well as in Penicillium swiecickii. Black aspergilli are cosmopolitan opportunistic fungi that can cause diverse mycoses and acute aspergillosis in immunocompromised individuals. Amongst the approximately 75 genome-sequenced A. niger strains, aoxB features considerable sequence variation. Five mutations were identified that rationally affect transcription or function or terminally modify the gene product. One mutant allele that occurs in CBS 513.88 and A. niger neotype strain CBS 554.65 involves a chromosomal deletion that removes exon 1 and intron 1 from aoxB. Another aoxB allele results from retrotransposon integration. Three other alleles result from point mutations: a missense mutation of the start codon, a frameshift, and a nonsense mutation. A. niger strain ATCC 1015 has a full-length aoxB gene. The A. niger sensu stricto complex can thus be subdivided into six taxa according to extant aoxB allele, which may facilitate rapid and accurate identification of individual species Full article

(This article belongs to the Special Issue Physiology and Biotechnology of Aspergillus niger)

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11 pages, 744 KiB

Open AccessEditor’s ChoiceArticle

Short Tandem Repeat Genotyping and Antifungal Susceptibility Testing of Latin American Candida tropicalis Isolates

by Bram Spruijtenburg, Cynthea C. S. Z. Baqueiro, Arnaldo L. Colombo, Eelco F. J. Meijer, João N. de Almeida, Jr., Indira Berrio, Norma B. Fernández, Guilherme M. Chaves, Jacques F. Meis, Theun de Groot and on behalf of the Latin American Group for Investigating Candida Tropicalis Resistance

J. Fungi 2023, 9(2), 207; https://doi.org/10.3390/jof9020207 - 5 Feb 2023

Cited by 10 | Viewed by 2871

Abstract

Candida tropicalis is emerging as one of the most common Candida species causing opportunistic infections in Latin America. Outbreak events caused by C. tropicalis were reported, and antifungal resistant isolates are on the rise. In order to investigate population genomics and look into [...] Read more.

Candida tropicalis is emerging as one of the most common Candida species causing opportunistic infections in Latin America. Outbreak events caused by C. tropicalis were reported, and antifungal resistant isolates are on the rise. In order to investigate population genomics and look into antifungal resistance, we applied a short tandem repeat (STR) genotyping scheme and antifungal susceptibility testing (AFST) to 230 clinical and environmental C. tropicalis isolates from Latin American countries. STR genotyping identified 164 genotypes, including 11 clusters comprised of three to seven isolates, indicating outbreak events. AFST identified one isolate as anidulafungin-resistant and harboring a FKS1 S659P substitution. Moreover, we identified 24 clinical and environmental isolates with intermediate susceptibility or resistance to one or more azoles. ERG11 sequencing revealed each of these isolates harboring a Y132F and/or Y257H/N substitution. All of these isolates, except one, were clustered together in two groups of closely related STR genotypes, with each group harboring distinct ERG11 substitutions. The ancestral C. tropicalis strain of these isolates likely acquired the azole resistance-associated substitutions and subsequently spread across vast distances within Brazil. Altogether, this STR genotyping scheme for C. tropicalis proved to be useful for identifying unrecognized outbreak events and better understanding population genomics, including the spread of antifungal-resistant isolates. Full article

(This article belongs to the Special Issue Pathogenicity and Molecular Biology of Human Pathogenic Fungi)

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16 pages, 1786 KiB

Open AccessEditor’s ChoiceArticle

Functional Expression of Recombinant Candida auris Proteins in Saccharomyces cerevisiae Enables Azole Susceptibility Evaluation and Drug Discovery

by Stephanie Toepfer, Michaela Lackner, Mikhail V. Keniya and Brian C. Monk

J. Fungi 2023, 9(2), 168; https://doi.org/10.3390/jof9020168 - 27 Jan 2023

Cited by 8 | Viewed by 3241

Abstract

Candida auris infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in C. auris are overexpression and point mutations in Erg11, and the overexpression of efflux pump genes CDR1 and [...] Read more.

Candida auris infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in C. auris are overexpression and point mutations in Erg11, and the overexpression of efflux pump genes CDR1 and MDR1. We report the establishment of a novel platform for molecular analysis and drug screening based on acquired azole-resistance mechanisms found in C. auris. Constitutive functional overexpression of wild-type C. auris Erg11, Erg11 with amino acid substitutions Y132F or K143R and the recombinant efflux pumps Cdr1 and Mdr1 has been achieved in Saccharomyces cerevisiae. Phenotypes were evaluated for standard azoles and the tetrazole VT-1161. Overexpression of CauErg11 Y132F, CauErg11 K143R, and CauMdr1 conferred resistance exclusively to the short-tailed azoles Fluconazole and Voriconazole. Strains overexpressing the Cdr1 protein were pan-azole resistant. While CauErg11 Y132F increased VT-1161 resistance, K143R had no impact. Type II binding spectra showed tight azole binding to the affinity-purified recombinant CauErg11 protein. The Nile Red assay confirmed the efflux functions of CauMdr1 and CauCdr1, which were specifically inhibited by MCC1189 and Beauvericin, respectively. CauCdr1 exhibited ATPase activity that was inhibited by Oligomycin. The S. cerevisiae overexpression platform enables evaluation of the interaction of existing and novel azole drugs with their primary target CauErg11 and their susceptibility to drug efflux. Full article

(This article belongs to the Special Issue Protein Engineering of Yeast)

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16 pages, 2315 KiB

Open AccessEditor’s ChoiceArticle

Volatile Metabolites in Lavage Fluid Are Correlated with Cytokine Production in a Valley Fever Murine Model

by Emily A. Higgins Keppler, Marley C. Caballero Van Dyke, Heather L. Mead, Douglas F. Lake, D. Mitchell Magee, Bridget M. Barker and Heather D. Bean

J. Fungi 2023, 9(1), 115; https://doi.org/10.3390/jof9010115 - 14 Jan 2023

Cited by 6 | Viewed by 2735

Abstract

Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi of arid regions in North and South America that are responsible for Valley fever (coccidioidomycosis). Forty percent of patients with Valley fever exhibit symptoms ranging from mild, self-limiting respiratory infections to severe, life-threatening pneumonia that [...] Read more.

Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi of arid regions in North and South America that are responsible for Valley fever (coccidioidomycosis). Forty percent of patients with Valley fever exhibit symptoms ranging from mild, self-limiting respiratory infections to severe, life-threatening pneumonia that requires treatment. Misdiagnosis as bacterial pneumonia commonly occurs in symptomatic Valley fever cases, resulting in inappropriate treatment with antibiotics, increased medical costs, and delay in diagnosis. In this proof-of-concept study, we explored the feasibility of developing breath-based diagnostics for Valley fever using a murine lung infection model. To investigate potential volatile biomarkers of Valley fever that arise from host–pathogen interactions, we infected C57BL/6J mice with C. immitis RS (n = 6), C. posadasii Silveira (n = 6), or phosphate-buffered saline (n = 4) via intranasal inoculation. We measured fungal dissemination and collected bronchoalveolar lavage fluid (BALF) for cytokine profiling and for untargeted volatile metabolomics via solid-phase microextraction (SPME) and two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS). We identified 36 volatile organic compounds (VOCs) that were significantly correlated (p < 0.05) with cytokine abundance. These 36 VOCs clustered mice by their cytokine production and were also able to separate mice with moderate-to-high cytokine production by infection strain. The data presented here show that Coccidioides and/or the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test that can detect coccidioidal infection and provide clinically relevant information on primary pulmonary disease severity. Full article

(This article belongs to the Special Issue Basic and Clinical Research on Coccidioides)

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18 pages, 4522 KiB

Open AccessEditor’s ChoiceArticle

Inhibition of Aflatoxin Production by Citrinin and Non-Enzymatic Formation of a Novel Citrinin-Kojic Acid Adduct

by Masayuki Ichinomiya, Emi Fukushima-Sakuno, Ayaka Kawamoto, Hiroyuki Nakagawa, Hidemi Hatabayashi, Hiromitsu Nakajima and Kimiko Yabe

J. Fungi 2023, 9(1), 29; https://doi.org/10.3390/jof9010029 - 23 Dec 2022

Cited by 3 | Viewed by 3454

Abstract

Screening for microorganisms that inhibit aflatoxin production from environments showed that Penicillium citrinum inhibited aflatoxin production by Aspergillus parasiticus. The inhibitory substance in the culture medium of P. citrinum was confirmed to be citrinin (CTN). RT-PCR analyses showed that CTN did not [...] Read more.

Screening for microorganisms that inhibit aflatoxin production from environments showed that Penicillium citrinum inhibited aflatoxin production by Aspergillus parasiticus. The inhibitory substance in the culture medium of P. citrinum was confirmed to be citrinin (CTN). RT-PCR analyses showed that CTN did not inhibit expressions of aflatoxin biosynthetic genes (aflR, pksL1, and fas-1) of A. parasiticus, whereas feeding experiments using A. parasiticus showed that CTN inhibited the in vivo conversion of dihydrosterigmatocystin to AFB₂·AFG₂. These results suggest that CTN inhibits a certain post-transcriptional step in aflatoxin biosynthesis. CTN in the culture medium of A. parasiticus was found to be decreased or lost with time, suggesting that a certain metabolite produced by A. parasiticus is the cause of the CTN decrease; we then purified, characterized, and then analyzed the substance. Physico-chemical analyses confirmed that the metabolite causing a decrease in CTN fluorescence was kojic acid (KA) and the resulting product was identified as a novel substance: (1R,3S,4R)-3,4-dihydro-6,8-dihydroxy-1-(3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)-3,4,5-trimethyl-1H-isochromene-7-carboxylic acid, which was named “CTN-KA adduct”. Our examination of the metabolites’ toxicities revealed that unlike CTN, the CTN-KA adduct did not inhibit aflatoxin production by A. parasiticus. These results indicate that CTN’s toxicity was alleviated with KA by converting CTN to the CTN-KA adduct. Full article

(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

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21 pages, 1370 KiB

Open AccessEditor’s ChoiceArticle

Antiproliferative Fatty Acids Isolated from the Polypore Fungus Onnia tomentosa

by Hooi Xian Lee, Wai Ming Li, Jatinder Khatra, Zhicheng Xia, Oleg Sannikov, Yun Ling, Haoxuan Zhu and Chow H. Lee

J. Fungi 2022, 8(11), 1163; https://doi.org/10.3390/jof8111163 - 3 Nov 2022

Viewed by 2339

Abstract

Onnia tomentosa is a widespread root rot pathogen frequently found in coniferous forests in North America. In this study, the potential medicinal properties of this wild polypore mushroom collected from north–central British Columbia, Canada, were investigated. The ethanol extract from O. tomentosa was [...] Read more.

Onnia tomentosa is a widespread root rot pathogen frequently found in coniferous forests in North America. In this study, the potential medicinal properties of this wild polypore mushroom collected from north–central British Columbia, Canada, were investigated. The ethanol extract from O. tomentosa was found to exhibit strong antiproliferative activity. Liquid–liquid extraction and bioactivity-guided fractionation, together with HPLC-MS/MS and 1D/2D NMR analyses of the ethanol extract of O. tomentosa, led to the identification of eight known linoleic oxygenated fatty acids (1.1–1.4 and 2–5), together with linoleic (6) and oleic acids (7). The autoxidation of linoleic acid upon isolation from a natural source and compound 5 as an autoxidation product of linoleic acid are reported here for the first time. GC-FID analysis of O. tomentosa, Fomitopsis officinalis, Echinodontium tinctorium, and Albatrellus flettii revealed linoleic, oleic, palmitic, and stearic acids as the major fatty acids. This study further showed that fatty acids were the major antiproliferative constituents in the ethanol extract from O. tomentosa. Linoleic acid and oleic acid had IC₅₀ values of 50.3 and 90.4 µM against human cervical cancer cells (HeLa), respectively. The results from this study have implications regarding the future exploration of O. tomentosa as a possible edible and/or medicinal mushroom. It is also recommended that necessary caution be taken when isolating unstable fatty acids from natural sources and in interpreting the results. Full article

(This article belongs to the Special Issue New Perspectives on Bioactive Compounds in Mushrooms)

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29 pages, 6682 KiB

Open AccessEditor’s ChoiceArticle

The Epichloë festucae Antifungal Protein Efe-AfpA Protects Creeping Bentgrass (Agrostis stolonifera) from the Plant Pathogen Clarireedia jacksonii, the Causal Agent of Dollar Spot Disease

by Patrick A. Fardella, Zipeng Tian, Bruce B. Clarke and Faith C. Belanger

J. Fungi 2022, 8(10), 1097; https://doi.org/10.3390/jof8101097 - 18 Oct 2022

Cited by 5 | Viewed by 2413

Abstract

Dollar spot disease, caused by the fungal pathogen Clarireedia jacksonii, is a major problem in many turfgrass species, particularly creeping bentgrass (Agrostis stolonifera). It is well-established that strong creeping red fescue (Festuca rubra subsp. rubra) exhibits good dollar [...] Read more.

Dollar spot disease, caused by the fungal pathogen Clarireedia jacksonii, is a major problem in many turfgrass species, particularly creeping bentgrass (Agrostis stolonifera). It is well-established that strong creeping red fescue (Festuca rubra subsp. rubra) exhibits good dollar spot resistance when infected by the fungal endophyte Epichloë festucae. This endophyte-mediated disease resistance is unique to the fine fescues and has not been observed in other grass species infected with other Epichloë spp. The mechanism underlying the unique endophyte-mediated disease resistance in strong creeping red fescue has not yet been established. We pursued the possibility that it may be due to the presence of an abundant secreted antifungal protein produced by E. festucae. Here, we compare the activity of the antifungal protein expressed in Escherichia coli, Pichia pastoris, and Penicillium chrysogenum. Active protein was recovered from all systems, with the best activity being from Pe. chrysogenum. In greenhouse assays, topical application of the purified antifungal protein to creeping bentgrass and endophyte-free strong creeping red fescue protected the plants from developing severe symptoms caused by C. jacksonii. These results support the hypothesis that Efe-AfpA is a major contributor to the dollar spot resistance observed with E. festucae-infected strong creeping red fescue in the field, and that this protein could be developed as an alternative or complement to fungicides for the management of this disease on turfgrasses. Full article

(This article belongs to the Special Issue Fungal Endophytes of Grasses)

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23 pages, 11957 KiB

Open AccessEditor’s ChoiceArticle

Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in Komagataella phaffii: A Budding Yeast in Distress

by Veronika Schusterbauer, Jasmin E. Fischer, Sarah Gangl, Lisa Schenzle, Claudia Rinnofner, Martina Geier, Christian Sailer, Anton Glieder and Gerhard G. Thallinger

J. Fungi 2022, 8(10), 992; https://doi.org/10.3390/jof8100992 - 21 Sep 2022

Cited by 9 | Viewed by 3554

Abstract

The industrially important non-conventional yeast Komagataella phaffii suffers from low rates of homologous recombination, making site specific genetic engineering tedious. Therefore, genome editing using CRISPR/Cas represents a simple and efficient alternative. To characterize on- and off-target mutations caused by CRISPR/Cas9 followed by non-homologous [...] Read more.

The industrially important non-conventional yeast Komagataella phaffii suffers from low rates of homologous recombination, making site specific genetic engineering tedious. Therefore, genome editing using CRISPR/Cas represents a simple and efficient alternative. To characterize on- and off-target mutations caused by CRISPR/Cas9 followed by non-homologous end joining repair, we chose a diverse set of CRISPR/Cas targets and conducted whole genome sequencing on 146 CRISPR/Cas9 engineered single colonies. We compared the outcomes of single target CRISPR transformations to double target experiments. Furthermore, we examined the extent of possible large deletions by targeting a large genomic region, which is likely to be non-essential. The analysis of on-target mutations showed an unexpectedly high number of large deletions and chromosomal rearrangements at the CRISPR target loci. We also observed an increase of on-target structural variants in double target experiments as compared to single target experiments. Targeting of two loci within a putatively non-essential region led to a truncation of chromosome 3 at the target locus in multiple cases, causing the deletion of 20 genes and several ribosomal DNA repeats. The identified de novo off-target mutations were rare and randomly distributed, with no apparent connection to unspecific CRISPR/Cas9 off-target binding sites. Full article

(This article belongs to the Special Issue Yeast Genetics 2022)

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18 pages, 2517 KiB

Open AccessEditor’s ChoiceArticle

Root Colonization by Fungal Entomopathogen Systemically Primes Belowground Plant Defense against Cabbage Root Fly

by Catalina Posada-Vergara, Katharina Lohaus, Mohammad Alhussein, Stefan Vidal and Michael Rostás

J. Fungi 2022, 8(9), 969; https://doi.org/10.3390/jof8090969 - 16 Sep 2022

Cited by 10 | Viewed by 3349

Abstract

Entomopathogenic fungi infect insects via spores but also live inside plant tissues as endophytes. Frequently, colonization by entomopathogens provides plants with increased resistance against insects, but the mechanisms are little understood. This study investigated direct, local, and systemic root-mediated interactions between isolates of [...] Read more.

Entomopathogenic fungi infect insects via spores but also live inside plant tissues as endophytes. Frequently, colonization by entomopathogens provides plants with increased resistance against insects, but the mechanisms are little understood. This study investigated direct, local, and systemic root-mediated interactions between isolates of the fungus Metarhizium brunneum and larvae of the cabbage root fly (CRF) Delia radicum attacking Brassica napus plants. All fungal isolates infected CRF when conidia were present in the soil, leading to 43–93% mortality. Locally, root-associated M. brunneum isolates reduced herbivore damage by 10–20% and in three out of five isolates caused significant insect mortality due to plant-mediated and/or direct effects. A split-root experiment with isolate Gd12 also demonstrated systemic plant resistance with significantly reduced root collar damage by CRF. LC-MS analyses showed that fungal root colonization did not induce changes in phytohormones, while herbivory increased jasmonic acid (JA) and glucosinolate concentrations. Proteinase inhibitor gene expression was also increased. Fungal colonization, however, primed herbivore-induced JA and the expression of the JA-responsive plant defensin 1.2 (PDF1.2) gene. We conclude that root-associated M. brunneum benefits plant health through multiple mechanisms, such as the direct infection of insects, as well as the local and systemic priming of the JA pathway. Full article

(This article belongs to the Special Issue Entomopathogenic Fungi in Biological Plant Protection: The Machinery of Multicomponent System Interactions)

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25 pages, 16080 KiB

Open AccessEditor’s ChoiceArticle

Occurrence, Diversity and Anti-Fungal Resistance of Fungi in Sand of an Urban Beach in Slovenia—Environmental Monitoring with Possible Health Risk Implications

by Monika Novak Babič, Nina Gunde-Cimerman, Martin Breskvar, Sašo Džeroski and João Brandão

J. Fungi 2022, 8(8), 860; https://doi.org/10.3390/jof8080860 - 16 Aug 2022

Cited by 12 | Viewed by 3785

Abstract

Beach safety regulation is based on faecal indicators in water, leaving out sand and fungi, whose presence in both matrices has often been reported. To study the abundance, diversity and possible fluctuations of mycobiota, fungi from sand and seawater were isolated from the [...] Read more.

Beach safety regulation is based on faecal indicators in water, leaving out sand and fungi, whose presence in both matrices has often been reported. To study the abundance, diversity and possible fluctuations of mycobiota, fungi from sand and seawater were isolated from the Portorož beach (Slovenia) during a 1-year period. Sand analyses yielded 64 species of 43 genera, whereas seawater samples yielded 29 species of 18 genera. Environmental and taxonomical data of fungal communities were analysed using machine learning approaches. Changes in the air and water temperature, sunshine hours, humidity and precipitation, air pressure and wind speed appeared to affect mycobiota. The core genera Aphanoascus, Aspergillus, Fusarium, Bisifusarium, Penicillium, Talaromyces, and Rhizopus were found to compose a stable community within sand, although their presence and abundance fluctuated along with weather changes. Aspergillus spp. were the most abundant and thus tested against nine antimycotics using Sensititre Yeast One kit. Aspergillus niger and A. welwitschiae isolates were found to be resistant to amphotericin B. Additionally, four possible human pollution indicators were isolated during the bathing season, including Meyerozyma, which can be used in beach microbial regulation. Our findings provide the foundations for additional research on sand and seawater mycobiota and show the potential effect of global warming and extreme weather events on fungi in sand and sea. Full article

(This article belongs to the Special Issue Fungal Diversity in Europe)

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20 pages, 2642 KiB

Open AccessEditor’s ChoiceArticle

The Dicarboxylate Transporters from the AceTr Family and Dct-02 Oppositely Affect Succinic Acid Production in S. cerevisiae

by Toni Rendulić, Frederico Mendonça Bahia, Isabel Soares-Silva, Elke Nevoigt and Margarida Casal

J. Fungi 2022, 8(8), 822; https://doi.org/10.3390/jof8080822 - 6 Aug 2022

Cited by 9 | Viewed by 3745

Abstract

Membrane transporters are important targets in metabolic engineering to establish and improve the production of chemicals such as succinic acid from renewable resources by microbial cell factories. We recently provided a Saccharomyces cerevisiae strain able to strongly overproduce succinic acid from glycerol and [...] Read more.

Membrane transporters are important targets in metabolic engineering to establish and improve the production of chemicals such as succinic acid from renewable resources by microbial cell factories. We recently provided a Saccharomyces cerevisiae strain able to strongly overproduce succinic acid from glycerol and CO₂ in which the Dct-02 transporter from Aspergillus niger, assumed to be an anion channel, was used to export succinic acid from the cells. In a different study, we reported a new group of succinic acid transporters from the AceTr family, which were also described as anion channels. Here, we expressed these transporters in a succinic acid overproducing strain and compared their impact on extracellular succinic acid accumulation with that of the Dct-02 transporter. The results show that the tested transporters of the AceTr family hinder succinic acid accumulation in the extracellular medium at low pH, which is in strong contrast to Dct-02. Data suggests that the AceTr transporters prefer monovalent succinate, whereas Dct-02 prefers divalent succinate anions. In addition, the results provided deeper insights into the characteristics of Dct-02, showing its ability to act as a succinic acid importer (thus being bidirectional) and verifying its capability of exporting malate. Full article

(This article belongs to the Special Issue Fungi Nutrient Transportation)

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17 pages, 4741 KiB

Open AccessEditor’s ChoiceArticle

Microbiomic Analysis of Bacteria Associated with Rock Tripe Lichens in Continental and Maritime Antarctic Regions

by Zichen He, Takeshi Naganuma, Ryosuke Nakai, Satoshi Imura, Megumu Tsujimoto and Peter Convey

J. Fungi 2022, 8(8), 817; https://doi.org/10.3390/jof8080817 - 3 Aug 2022

Cited by 8 | Viewed by 4919

Abstract

Increased research attention is being given to bacterial diversity associated with lichens. Rock tripe lichens (Umbilicariaceae) were collected from two distinct Antarctic biological regions, the continental region near the Japanese Antarctic station (Syowa Station) and the maritime Antarctic South Orkney Islands [...] Read more.

Increased research attention is being given to bacterial diversity associated with lichens. Rock tripe lichens (Umbilicariaceae) were collected from two distinct Antarctic biological regions, the continental region near the Japanese Antarctic station (Syowa Station) and the maritime Antarctic South Orkney Islands (Signy Island), in order to compare their bacterial floras and potential metabolism. Bulk DNA extracted from the lichen samples was used to amplify the 18S rRNA gene and the V3-V4 region of the 16S rRNA gene, whose amplicons were Sanger- and MiSeq-sequenced, respectively. The fungal and algal partners represented members of the ascomycete genus Umbilicaria and the green algal genus Trebouxia, based on 18S rRNA gene sequences. The V3-V4 sequences were grouped into operational taxonomic units (OTUs), which were assigned to eight bacterial phyla, Acidobacteriota, Actinomyceota, Armatimonadota, Bacteroidota, Cyanobacteria, Deinococcota, Pseudomonadota and the candidate phylum Saccharibacteria (also known as TM7), commonly present in all samples. The OTU floras of the two biological regions were clearly distinct, with regional biomarker genera, such as Mucilaginibacter and Gluconacetobacter, respectively. The OTU-based metabolism analysis predicted higher membrane transport activities in the maritime Antarctic OTUs, probably influenced by the sampling area’s warmer maritime climatic setting. Full article

(This article belongs to the Special Issue Ecology and Evolution of Lichens and Associated Microorganisms)

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16 pages, 4245 KiB

Open AccessEditor’s ChoiceArticle

The Fungal Protein Mes1 Is Required for Morphogenesis and Virulence in the Dimorphic Phytopathogen Ustilago maydis

by David Cánovas

J. Fungi 2022, 8(8), 759; https://doi.org/10.3390/jof8080759 - 22 Jul 2022

Cited by 3 | Viewed by 2802

Abstract

Polarized growth is a defining property of filamentous fungi, which plays an important role in different aspects of their biology, including virulence. However, little information is available about the determinants of cell surface organization and their role in polarized growth. The fungal protein [...] Read more.

Polarized growth is a defining property of filamentous fungi, which plays an important role in different aspects of their biology, including virulence. However, little information is available about the determinants of cell surface organization and their role in polarized growth. The fungal protein MesA was identified in a genetic screen in Aspergillus nidulans and is involved in the stabilization of the polarity axes, but it has no evident role in budding yeast. In this work, I present evidence that in the dimorphic fungal phytopathogen Ustilago maydis MesA/Mes1 is involved in cell wall stability and polarized growth. mes1 mutants were more sensitive to drugs provoking cell wall stress, and they displayed a temperature-sensitive phenotype. Actin cytoskeleton was disorganized in a mes1 mutant, suggesting that there is a connection between Mes1, the actin cytoskeleton and polarized morphogenesis. The septin ring was also absent from the bud tip, but not the bud neck. Deletion of mes1 provoked defects in endocytosis and vacuolar organization in the cells. Mes1 was essential for strong polarized growth in the hyphal form, but it was dispensable during low or moderate polarized growth in the yeast form in U. maydis at a permissive temperature. Consistently, mes1 mutants showed delayed mating and they were avirulent. Full article

(This article belongs to the Special Issue Interactions between Filamentous Fungal Pathogens and Hosts)

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21 pages, 1409 KiB

Open AccessEditor’s ChoiceArticle

Fungal Community Composition as Affected by Litter Chemistry and Weather during Four Years of Litter Decomposition in Rainshadow Coastal Douglas-Fir Forests

by Philip-Edouard Shay, Richard S. Winder, C. Peter Constabel and J. A. (Tony) Trofymow

J. Fungi 2022, 8(7), 735; https://doi.org/10.3390/jof8070735 - 16 Jul 2022

Viewed by 2399

Abstract

Climate and litter chemistry are major factors influencing litter decay, a process mediated by microbes, such as fungi, nitrogen-fixing bacteria and ammonia-oxidizing bacteria. Increasing atmospheric CO₂ concentrations can decrease nitrogen (N) and increase condensed tannin (CT) content in foliar litter, reducing litter [...] Read more.

Climate and litter chemistry are major factors influencing litter decay, a process mediated by microbes, such as fungi, nitrogen-fixing bacteria and ammonia-oxidizing bacteria. Increasing atmospheric CO₂ concentrations can decrease nitrogen (N) and increase condensed tannin (CT) content in foliar litter, reducing litter quality and slowing decomposition. We hypothesized that reduced litter quality inhibits microbes and is the mechanism causing decomposition to slow. Litterbags of Douglas-fir needles and poplar leaves with a range of N (0.61–1.57%) and CT (2.1–29.1%) treatment and natural acid unhydrolyzable residue (35.3–41.5%) concentrations were placed along climatic gradients in mature Douglas-fir stands of coastal British Columbia rainshadow forests. The structure (diversity, richness and evenness) and composition of microbial communities were analyzed using DGGE profiles of 18S, NifH-universal and AmoA PCR amplicons in foliar litter after 7, 12, 24 and 43 months of decay. High CT and low N concentrations in leaf litter were associated with changes in microbial community composition, especially fungi. Contrary to our hypothesis, high CT and low N treatments did not inhibit microbial colonization or diversity. The joint effects of air temperature and soil moisture on microbial community composition at our sites were more important than the effects of initial litter chemistry. Full article

(This article belongs to the Special Issue Fungi in Decomposition Processes)

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25 pages, 4014 KiB

Open AccessEditor’s ChoiceArticle

Nitric Oxide Metabolism Affects Germination in Botrytis cinerea and Is Connected to Nitrate Assimilation

by Francisco Anta-Fernández, Daniela Santander-Gordón, Sioly Becerra, Rodrigo Santamaría, José María Díaz-Mínguez and Ernesto Pérez Benito

J. Fungi 2022, 8(7), 699; https://doi.org/10.3390/jof8070699 - 1 Jul 2022

Cited by 4 | Viewed by 4327

Abstract

Nitric oxide regulates numerous physiological processes in species from all taxonomic groups. Here, its role in the early developmental stages of the fungal necrotroph Botrytis cinerea was investigated. Pharmacological analysis demonstrated that NO modulated germination, germ tube elongation and nuclear division rate. Experimental [...] Read more.

Nitric oxide regulates numerous physiological processes in species from all taxonomic groups. Here, its role in the early developmental stages of the fungal necrotroph Botrytis cinerea was investigated. Pharmacological analysis demonstrated that NO modulated germination, germ tube elongation and nuclear division rate. Experimental evidence indicates that exogenous NO exerts an immediate but transitory negative effect, slowing down germination-associated processes, and that this effect is largely dependent on the flavohemoglobin BCFHG1. The fungus exhibited a “biphasic response” to NO, being more sensitive to low and high concentrations than to intermediate levels of the NO donor. Global gene expression analysis in the wild-type and ΔBcfhg1 strains indicated a situation of strong nitrosative and oxidative stress determined by exogenous NO, which was much more intense in the mutant strain, that the cells tried to alleviate by upregulating several defense mechanisms, including the simultaneous upregulation of the genes encoding the flavohemoglobin BCFHG1, a nitronate monooxygenase (NMO) and a cyanide hydratase. Genetic evidence suggests the coordinated expression of Bcfhg1 and the NMO coding gene, both adjacent and divergently arranged, in response to NO. Nitrate assimilation genes were upregulated upon exposure to NO, and BCFHG1 appeared to be the main enzymatic system involved in the generation of the signal triggering their induction. Comparative expression analysis also showed the influence of NO on other cellular processes, such as mitochondrial respiration or primary and secondary metabolism, whose response could have been mediated by NmrA-like domain proteins. Full article

(This article belongs to the Special Issue Infection Mechanisms of Botrytis cinerea and Compounds with Antifungal Activity)

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10 pages, 1992 KiB

Open AccessEditor’s ChoiceArticle

pH Distribution along Growing Fungal Hyphae at Microscale

by Bi-Jing Xiong, Claire E. Stanley, Christian Dusny, Dietmar Schlosser, Hauke Harms and Lukas Y. Wick

J. Fungi 2022, 8(6), 599; https://doi.org/10.3390/jof8060599 - 3 Jun 2022

Cited by 4 | Viewed by 3548

Abstract

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing [...] Read more.

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering. Full article

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14 pages, 5875 KiB

Open AccessEditor’s ChoiceArticle

Identification and Functional Characterization of a Putative Alternative Oxidase (Aox) in Sporisorium reilianum f. sp. zeae

by Hector Mendoza, Caroline D. Culver, Emma A. Lamb, Luke A. Schroeder, Sunita Khanal, Christian Müller, Jan Schirawski and Michael H. Perlin

J. Fungi 2022, 8(2), 148; https://doi.org/10.3390/jof8020148 - 31 Jan 2022

Cited by 3 | Viewed by 4234

Abstract

The mitochondrial electron transport chain consists of the classical protein complexes (I–IV) that facilitate the flow of electrons and coupled oxidative phosphorylation to produce metabolic energy. The canonical route of electron transport may diverge by the presence of alternative components to the electron [...] Read more.

The mitochondrial electron transport chain consists of the classical protein complexes (I–IV) that facilitate the flow of electrons and coupled oxidative phosphorylation to produce metabolic energy. The canonical route of electron transport may diverge by the presence of alternative components to the electron transport chain. The following study comprises the bioinformatic identification and functional characterization of a putative alternative oxidase in the smut fungus Sporisorium reilianum f. sp. zeae. This alternative respiratory component has been previously identified in other eukaryotes and is essential for alternative respiration as a response to environmental and chemical stressors, as well as for developmental transitionaoxs during the life cycle of an organism. A growth inhibition assay, using specific mitochondrial inhibitors, functionally confirmed the presence of an antimycin-resistant/salicylhydroxamic acid (SHAM)-sensitive alternative oxidase in the respirasome of S. reilianum. Gene disruption experiments revealed that this enzyme is involved in the pathogenic stage of the fungus, with its absence effectively reducing overall disease incidence in infected maize plants. Furthermore, gene expression analysis revealed that alternative oxidase plays a prominent role in the teliospore developmental stage, in agreement with favoring alternative respiration during quiescent stages of an organism’s life cycle. Full article

(This article belongs to the Special Issue Smut Fungi 2.0)

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33 pages, 6209 KiB

Open AccessEditor’s ChoiceArticle

Reappraisal of the Genus Exsudoporus (Boletaceae) Worldwide Based on Multi-Gene Phylogeny, Morphology and Biogeography, and Insights on Amoenoboletus

by Alona Yu. Biketova, Matteo Gelardi, Matthew E. Smith, Giampaolo Simonini, Rosanne A. Healy, Yuichi Taneyama, Gianrico Vasquez, Ádám Kovács, László G. Nagy, Solomon P. Wasser, Ursula Peintner, Eviatar Nevo, Britt A. Bunyard and Alfredo Vizzini

J. Fungi 2022, 8(2), 101; https://doi.org/10.3390/jof8020101 - 21 Jan 2022

Cited by 9 | Viewed by 7470

Abstract

The boletoid genera Butyriboletus and Exsudoporus have recently been suggested by some researchers to constitute a single genus, and Exsudoporus was merged into Butyriboletus as a later synonym. However, no convincing arguments have yet provided significant evidence for this congeneric placement. In this [...] Read more.

The boletoid genera Butyriboletus and Exsudoporus have recently been suggested by some researchers to constitute a single genus, and Exsudoporus was merged into Butyriboletus as a later synonym. However, no convincing arguments have yet provided significant evidence for this congeneric placement. In this study, we analyze material from Exsudoporus species and closely related taxa to assess taxonomic and phylogenetic boundaries between these genera and to clarify species delimitation within Exsudoporus. Outcomes from a multilocus phylogenetic analysis (ITS, nrLSU, tef1-α and rpb2) clearly resolve Exsudoporus as a monophyletic, homogenous and independent genus that is sister to Butyriboletus. An accurate morphological description, comprehensive sampling, type studies, line drawings and a historical overview on the nomenclatural issues of the type species E. permagnificus are provided. Furthermore, this species is documented for the first time from Israel in association with Quercus calliprinos. The previously described North American species Exsudoporus frostii and E. floridanus are molecularly confirmed as representatives of Exsudoporus, and E. floridanus is epitypified. The eastern Asian species Leccinum rubrum is assigned here to Exsudoporus based on molecular evidence, and a new combination is proposed. Sequence data from the original material of the Japanese Boletus kermesinus were generated, and its conspecificity with L. rubrum is inferred as formerly presumed based on morphology. Four additional cryptic species from North and Central America previously misdetermined as either B. frostii or B. floridanus are phylogenetically placed but remain undescribed due to the paucity of available material. Boletus weberi (syn. B. pseudofrostii) and Xerocomus cf. mcrobbii cluster outside of Exsudoporus and are herein assigned to the recently described genus Amoenoboletus. Biogeographic distribution patterns are elucidated, and a dichotomous key to all known species of Exsudoporus worldwide is presented. Full article

(This article belongs to the Special Issue Biodiversity, Distribution and Conservation of Plants and Fungi; Effects of Global Warming and Environmental Stress)

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20 pages, 4411 KiB

Open AccessEditor’s ChoiceArticle

Characterisation of Candida parapsilosis CYP51 as a Drug Target Using Saccharomyces cerevisiae as Host

by Yasmeen N. Ruma, Mikhail V. Keniya, Joel D. A. Tyndall and Brian C. Monk

J. Fungi 2022, 8(1), 69; https://doi.org/10.3390/jof8010069 - 10 Jan 2022

Cited by 13 | Viewed by 3481

Abstract

The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that [...] Read more.

The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs. Candida parapsilosis is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type C. parapsilosis CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a Saccharomyces cerevisiae host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 Å. Structural analysis of azole—enzyme interactions and functional studies of recombinant CYP51 from C. parapsilosis have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery. Full article

(This article belongs to the Special Issue Genetic Manipulation of Fungal Model Organisms)

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23 pages, 6108 KiB

Open AccessEditor’s ChoiceArticle

Uncovering Novel Plasma Membrane Carboxylate Transporters in the Yeast Cyberlindnera jadinii

by Maria Sousa-Silva, Pedro Soares, João Alves, Daniel Vieira, Margarida Casal and Isabel Soares-Silva

J. Fungi 2022, 8(1), 51; https://doi.org/10.3390/jof8010051 - 5 Jan 2022

Cited by 4 | Viewed by 3564

Abstract

The yeast Cyberlindnera jadinii has great potential in the biotechnology industry due to its ability to produce a variety of compounds of interest, including carboxylic acids. In this work, we identified genes encoding carboxylate transporters from this yeast species. The functional characterization of [...] Read more.

The yeast Cyberlindnera jadinii has great potential in the biotechnology industry due to its ability to produce a variety of compounds of interest, including carboxylic acids. In this work, we identified genes encoding carboxylate transporters from this yeast species. The functional characterization of sixteen plasma membrane carboxylate transporters belonging to the AceTr, SHS, TDT, MCT, SSS, and DASS families was performed by heterologous expression in Saccharomyces cerevisiae. The newly identified C. jadinii transporters present specificity for mono-, di-, and tricarboxylates. The transporters CjAto5, CjJen6, CjSlc5, and CjSlc13-1 display the broadest substrate specificity; CjAto2 accepts mono- and dicarboxylates; and CjAto1,3,4, CjJen1-5, CjSlc16, and CjSlc13-2 are specific for monocarboxylic acids. A detailed characterization of these transporters, including phylogenetic reconstruction, 3D structure prediction, and molecular docking analysis is presented here. The properties presented by these transporters make them interesting targets to be explored as organic acid exporters in microbial cell factories. Full article

(This article belongs to the Special Issue Fungi Nutrient Transportation)

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18 pages, 2943 KiB

Open AccessEditor’s ChoiceArticle

Engineering Aspergillus oryzae for the Heterologous Expression of a Bacterial Modular Polyketide Synthase

by Jin Feng, Maurice Hauser, Russell J. Cox and Elizabeth Skellam

J. Fungi 2021, 7(12), 1085; https://doi.org/10.3390/jof7121085 - 17 Dec 2021

Cited by 7 | Viewed by 5248

Abstract

Microbial natural products have had phenomenal success in drug discovery and development yet form distinct classes based on the origin of their native producer. Methods that enable metabolic engineers to combine the most useful features of the different classes of natural products may [...] Read more.

Microbial natural products have had phenomenal success in drug discovery and development yet form distinct classes based on the origin of their native producer. Methods that enable metabolic engineers to combine the most useful features of the different classes of natural products may lead to molecules with enhanced biological activities. In this study, we modified the metabolism of the fungus Aspergillus oryzae to enable the synthesis of triketide lactone (TKL), the product of the modular polyketide synthase DEBS1-TE engineered from bacteria. We established (2S)-methylmalonyl-CoA biosynthesis via introducing a propionyl-CoA carboxylase complex (PCC); reassembled the 11.2 kb DEBS1-TE coding region from synthetic codon-optimized gene fragments using yeast recombination; introduced bacterial phosphopantetheinyltransferase SePptII; investigated propionyl-CoA synthesis and degradation pathways; and developed improved delivery of exogenous propionate. Depending on the conditions used titers of TKL ranged from <0.01–7.4 mg/L. In conclusion, we have demonstrated that A. oryzae can be used as an alternative host for the synthesis of polyketides from bacteria, even those that require toxic or non-native substrates. Our metabolically engineered A. oryzae may offer advantages over current heterologous platforms for producing valuable and complex natural products. Full article

(This article belongs to the Special Issue Secondary Metabolites from Fungi—in Honour of Prof. Dr. Ji-Kai Liu's 60th Birthday)

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17 pages, 2492 KiB

Open AccessEditor’s ChoiceArticle

Bile Acid Regulates the Colonization and Dissemination of Candida albicans from the Gastrointestinal Tract by Controlling Host Defense System and Microbiota

by Shankar Thangamani, Ross Monasky, Jung Keun Lee, Vijay Antharam, Harm HogenEsch, Tony R. Hazbun, Yan Jin, Haiwei Gu and Grace L. Guo

J. Fungi 2021, 7(12), 1030; https://doi.org/10.3390/jof7121030 - 30 Nov 2021

Cited by 13 | Viewed by 4471

Abstract

Candida albicans (CA), a commensal and opportunistic eukaryotic organism, frequently inhabits the gastrointestinal (GI) tract and causes life-threatening infections. Antibiotic-induced gut dysbiosis is a major risk factor for increased CA colonization and dissemination from the GI tract. We identified a significant increase of [...] Read more.

Candida albicans (CA), a commensal and opportunistic eukaryotic organism, frequently inhabits the gastrointestinal (GI) tract and causes life-threatening infections. Antibiotic-induced gut dysbiosis is a major risk factor for increased CA colonization and dissemination from the GI tract. We identified a significant increase of taurocholic acid (TCA), a major bile acid in antibiotic-treated mice susceptible to CA infection. In vivo findings indicate that administration of TCA through drinking water is sufficient to induce colonization and dissemination of CA in wild-type and immunosuppressed mice. Treatment with TCA significantly reduced mRNA expression of immune genes ang4 and Cxcr3 in the colon. In addition, TCA significantly decreased the relative abundance of three culturable species of commensal bacteria, Turicibacter sanguinis, Lactobacillus johnsonii, and Clostridium celatum, in both cecal contents and mucosal scrapings from the colon. Taken together, our results indicate that TCA promotes fungal colonization and dissemination of CA from the GI tract by controlling the host defense system and intestinal microbiota that play a critical role in regulating CA in the intestine. Full article

(This article belongs to the Special Issue Systems Biology in Fungal Research)

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13 pages, 3145 KiB

Open AccessEditor’s ChoiceArticle

Novel Pathogenic Mucorales Identified Using the Silkworm Infection Model

by Suresh Panthee, Hiroshi Hamamoto, Yayoi Nishiyama, Atmika Paudel and Kazuhisa Sekimizu

J. Fungi 2021, 7(11), 995; https://doi.org/10.3390/jof7110995 - 22 Nov 2021

Cited by 5 | Viewed by 4456

Abstract

Mucormycosis, a rare but highly fatal infection, is caused by fungi of the order Mucorales. Due to their ubiquitous nature, reduced susceptibility to antifungals, acid tolerance, and ability to infect immunocompromised patients through rapid dissemination, these fungi have been frequently reported to infect [...] Read more.

Mucormycosis, a rare but highly fatal infection, is caused by fungi of the order Mucorales. Due to their ubiquitous nature, reduced susceptibility to antifungals, acid tolerance, and ability to infect immunocompromised patients through rapid dissemination, these fungi have been frequently reported to infect the COVID-19 patients. In order to develop strategies to overcome mucormycosis, it is essential to understand and identify novel Mucorales present in the environment. In this study, we report the identification of four novel pathogenic Mucorales using the silkworm (Bombyx mori) model. The strains’ phylogeny was analyzed using the genome sequence of the large subunit ribosomal ribonucleic acid (LSU rRNA) and the internal transcribed spacer (ITS) region, where strains 1-3, 5-3, and S286-1101 claded with Mucor orantomantidis, and strain 827-14 claded with Backusella lamprospora. All the strains had a cold-sensitive phenotype with their inability to grow prominently at 4 °C. Mucor sp. 1-3 and 5-3 were characterized by their filamentous and yeast-like growth under aerobic and anaerobic conditions, respectively. The yeast colonies of Mucor sp. 5-3 had multipolar budding cells often observed with cleaved cell surfaces under a scanning electron microscope. We further found that these strains were able to kill immunocompromised mice suggesting their pathogenicity to mammals. Our study established an invertebrate model-based screening system to identify novel pathogenic Mucorales from the natural environment and provided a clue towards the rapid increase in COVID-19 related mucormycosis. Full article

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22 pages, 5289 KiB

Open AccessEditor’s ChoiceArticle

Phylogenetic and Functional Diversity of Saprolegniales and Fungi Isolated from Temperate Lakes in Northeast Germany

by Hossein Masigol, Jason Nicholas Woodhouse, Pieter van West, Reza Mostowfizadeh-Ghalamfarsa, Keilor Rojas-Jimenez, Tobias Goldhammer, Seyed Akbar Khodaparast and Hans-Peter Grossart

J. Fungi 2021, 7(11), 968; https://doi.org/10.3390/jof7110968 - 13 Nov 2021

Cited by 8 | Viewed by 3834

Abstract

The contribution of fungi to the degradation of plant litter and transformation of dissolved organic matter (humic substances, in particular) in freshwater ecosystems has received increasing attention recently. However, the role of Saprolegniales as one of the most common eukaryotic organisms is rarely [...] Read more.

The contribution of fungi to the degradation of plant litter and transformation of dissolved organic matter (humic substances, in particular) in freshwater ecosystems has received increasing attention recently. However, the role of Saprolegniales as one of the most common eukaryotic organisms is rarely studied. In this study, we isolated and phylogenetically placed 51 fungal and 62 Saprolegniales strains from 12 German lakes. We studied the cellulo-, lignino-, and chitinolytic activity of the strains using plate assays. Furthermore, we determined the capacity of 10 selected strains to utilize 95 different labile compounds, using Biolog FF MicroPlates™. Finally, the ability of three selected strains to utilize maltose and degrade/produce humic substances was measured. Cladosporium and Penicillium were amongst the most prevalent fungal strains, while Saprolegnia, Achlya, and Leptolegnia were the most frequent Saprolegniales strains. Although the isolated strains assigned to genera were phylogenetically similar, their enzymatic activity and physiological profiling were quite diverse. Our results indicate that Saprolegniales, in contrast to fungi, lack ligninolytic activity and are not involved in the production/transformation of humic substances. We hypothesize that Saprolegniales and fungi might have complementary roles in interacting with dissolved organic matter, which has ecological implications for carbon cycling in freshwater ecosystems. Full article

(This article belongs to the Special Issue Oomycetes – Diversity, Taxonomy, Evolution, Ecology and Host-Pathogen Interactions)

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16 pages, 2998 KiB

Open AccessEditor’s ChoiceArticle

P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity

by Lyubomir Dimitrov Stanchev, Juliana Rizzo, Rebecca Peschel, Lilli A. Pazurek, Lasse Bredegaard, Sarina Veit, Sabine Laerbusch, Marcio L. Rodrigues, Rosa L. López-Marqués and Thomas Günther Pomorski

J. Fungi 2021, 7(10), 843; https://doi.org/10.3390/jof7100843 - 8 Oct 2021

Cited by 8 | Viewed by 4642

Abstract

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but [...] Read more.

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development. Full article

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13 pages, 2330 KiB

Open AccessEditor’s ChoiceArticle

Unveiling the Role Displayed by Penicillium digitatum PdMut3 Transcription Factor in Pathogen–Fruit Interaction

by Marta de Ramón-Carbonell and Paloma Sánchez-Torres

J. Fungi 2021, 7(10), 828; https://doi.org/10.3390/jof7100828 - 3 Oct 2021

Cited by 9 | Viewed by 2806

Abstract

Zn₂Cys₆ transcription factors are unique to fungi and are involved in different regulatory functions. In this study, we have identified the Penicillium digitatumPdMut3 gene, which encodes a putative Zn (II) 2Cys6 DNA-binding protein. Elimination of PdMut3 in Pd1 strain [...] Read more.

Zn₂Cys₆ transcription factors are unique to fungi and are involved in different regulatory functions. In this study, we have identified the Penicillium digitatumPdMut3 gene, which encodes a putative Zn (II) 2Cys6 DNA-binding protein. Elimination of PdMut3 in Pd1 strain caused increased virulence during citrus infection. The transcription of the PdMut3 gene showed a higher expression rate during fungal growth and less transcription during fruit infection. Furthermore, the deletion of the gene in the wild-type isolate of P. digitatum did not produce any modification of the sensitivity to different fungicides, indicating that the gene is not associated with resistance to fungicides. In contrast, PdMut3 null mutants showed a reduction in growth in minimal media, which was associated with severe alterations in conidiophore development and morphological alterations of the hyphae. Mutants showed greater sensitivity to compounds that interfere with the cell wall and an invasive growth block. Thus, PdMut3 might have an indirect role in fungi virulence through metabolism and peroxisomes development. Full article

(This article belongs to the Special Issue Control of Postharvest Pathogenic Penicillium)

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13 pages, 1834 KiB

Open AccessEditor’s ChoiceCase Report

Noninvasive Combined Diagnosis and Monitoring of Aspergillus and Pseudomonas Infections: Proof of Concept

by Radim Dobiáš, Anton Škríba, Tomáš Pluháček, Miloš Petřík, Andrea Palyzová, Marcela Káňová, Eva Čubová, Jiří Houšť, Jiří Novák, David A. Stevens, Goran Mitulovič, Eva Krejčí, Petr Hubáček and Vladimír Havlíček

J. Fungi 2021, 7(9), 730; https://doi.org/10.3390/jof7090730 - 6 Sep 2021

Cited by 12 | Viewed by 3290

Abstract

In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient’s course. [...] Read more.

In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient’s course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections. Full article

(This article belongs to the Special Issue Metallophores in Diagnosing Infectious Diseases)

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21 pages, 5135 KiB

Open AccessEditor’s ChoiceArticle

Internally Symmetrical Stwintrons and Related Canonical Introns in Hypoxylaceae Species

by Erzsébet Fekete, Fruzsina Pénzes, Norbert Ág, Claudio Scazzocchio, Michel Flipphi and Levente Karaffa

J. Fungi 2021, 7(9), 710; https://doi.org/10.3390/jof7090710 - 29 Aug 2021

Cited by 2 | Viewed by 3017

Abstract

Spliceosomal introns are pervasive in eukaryotes. Intron gains and losses have occurred throughout evolution, but the origin of new introns is unclear. Stwintrons are complex intervening sequences where one of the sequence elements (5′-donor, lariat branch point element or 3′-acceptor) necessary for excision [...] Read more.

Spliceosomal introns are pervasive in eukaryotes. Intron gains and losses have occurred throughout evolution, but the origin of new introns is unclear. Stwintrons are complex intervening sequences where one of the sequence elements (5′-donor, lariat branch point element or 3′-acceptor) necessary for excision of a U2 intron (external intron) is itself interrupted by a second (internal) U2 intron. In Hypoxylaceae, a family of endophytic fungi, we uncovered scores of donor-disrupted stwintrons with striking sequence similarity among themselves and also with canonical introns. Intron–exon structure comparisons suggest that these stwintrons have proliferated within diverging taxa but also give rise to proliferating canonical introns in some genomes. The proliferated (stw)introns have integrated seamlessly at novel gene positions. The recently proliferated (stw)introns appear to originate from a conserved ancestral stwintron characterised by terminal inverted repeats (45–55 nucleotides), a highly symmetrical structure that may allow the formation of a double-stranded intron RNA molecule. No short tandem duplications flank the putatively inserted intervening sequences, which excludes a DNA transposition-based mechanism of proliferation. It is tempting to suggest that this highly symmetrical structure may have a role in intron proliferation by (an)other mechanism(s). Full article

(This article belongs to the Section Fungal Genomics, Genetics and Molecular Biology)

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16 pages, 8556 KiB

Open AccessEditor’s ChoiceArticle

Development of Carbazole Derivatives Compounds against Candida albicans: Candidates to Prevent Hyphal Formation via the Ras1-MAPK Pathway

by Young-Kwang Park, Jisoo Shin, Hee-Yoon Lee, Hag-Dong Kim and Joon Kim

J. Fungi 2021, 7(9), 688; https://doi.org/10.3390/jof7090688 - 25 Aug 2021

Cited by 8 | Viewed by 3241

Abstract

Morphogenesis contributes to the virulence of the opportunistic human fungal pathogen Candida albicans. Ras1-MAPK pathways play a critical role in the virulence of C. albicans by regulating cell growth, morphogenesis, and biofilm formation. Ume6 acts as a transcription factor, and Nrg1 is [...] Read more.

Morphogenesis contributes to the virulence of the opportunistic human fungal pathogen Candida albicans. Ras1-MAPK pathways play a critical role in the virulence of C. albicans by regulating cell growth, morphogenesis, and biofilm formation. Ume6 acts as a transcription factor, and Nrg1 is a transcriptional repressor for the expression of hyphal-specific genes in morphogenesis. Azoles or echinocandin drugs have been extensively prescribed for C. albicans infections, which has led to the development of drug-resistant strains. Therefore, it is necessary to develop new molecules to effectively treat fungal infections. Here, we showed that Molecule B and Molecule C, which contained a carbazole structure, attenuated the pathogenicity of C. albicans through inhibition of the Ras1/MAPK pathway. We found that Molecule B and Molecule C inhibit morphogenesis through repressing protein and RNA levels of Ras/MAPK-related genes, including UME6 and NRG1. Furthermore, we determined the antifungal effects of Molecule B and Molecule C in vivo using a candidiasis murine model. We anticipate our findings are that Molecule B and Molecule C, which inhibits the Ras1/MAPK pathway, are promising compounds for the development of new antifungal agents for the treatment of systemic candidiasis and possibly for other fungal diseases. Full article

(This article belongs to the Special Issue New Perspectives for Candidiasis)

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22 pages, 10857 KiB

Open AccessEditor’s ChoiceArticle

Drought Influences Fungal Community Dynamics in the Grapevine Rhizosphere and Root Microbiome

by María Julia Carbone, Sandra Alaniz, Pedro Mondino, Matías Gelabert, Ales Eichmeier, Dorota Tekielska, Rebeca Bujanda and David Gramaje

J. Fungi 2021, 7(9), 686; https://doi.org/10.3390/jof7090686 - 25 Aug 2021

Cited by 43 | Viewed by 6267

Abstract

Plant roots support complex microbial communities that can influence nutrition, plant growth, and health. In grapevine, little is known about the impact of abiotic stresses on the belowground microbiome. In this study, we examined the drought-induced shifts in fungal composition in the root [...] Read more.

Plant roots support complex microbial communities that can influence nutrition, plant growth, and health. In grapevine, little is known about the impact of abiotic stresses on the belowground microbiome. In this study, we examined the drought-induced shifts in fungal composition in the root endosphere, the rhizosphere and bulk soil by internal transcribed spacer (ITS) high-throughput amplicon sequencing (HTAS). We imposed three irrigation regimes (100%, 50%, and 25% of the field capacity) to one-year old grapevine rootstock plants cv. SO₄ when plants had developed 2–3 roots. Root endosphere, rhizosphere, and bulk soil samples were collected 6- and 12-months post-plantation. Drought significantly modified the overall fungal composition of all three compartments, with the root endosphere compartment showing the greatest divergence from well-watered control (100%). The overall response of the fungal microbiota associated with black-foot disease (Dactylonectria and “Cylindrocarpon” genera) and the potential biocontrol agent Trichoderma to drought stress was consistent across compartments, namely that their relative abundances were significantly higher at 50–100% than at 25% irrigation regime. We identified a significant enrichment in several fungal genera such as the arbuscular mycorrhizal fungus Funneliformis during drought at 25% watering regime within the roots. Our results reveal that drought stress, in addition to its well-characterized effects on plant physiology, also results in the restructuring of grapevine root microbial communities, and suggest the possibility that members of the altered grapevine microbiota might contribute to plant survival under extreme environmental conditions. Full article

(This article belongs to the Special Issue Application of Omics Technologies for a Deeper Insight into the Study of Fungal Trunk Pathogens of Grapevine)

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21 pages, 3529 KiB

Open AccessEditor’s ChoiceArticle

mRNA Inventory of Extracellular Vesicles from Ustilago maydis

by Seomun Kwon, Oliver Rupp, Andreas Brachmann, Christopher Frederik Blum, Anton Kraege, Alexander Goesmann and Michael Feldbrügge

J. Fungi 2021, 7(7), 562; https://doi.org/10.3390/jof7070562 - 14 Jul 2021

Cited by 37 | Viewed by 5736

Abstract

Extracellular vesicles (EVs) can transfer diverse RNA cargo for intercellular communication. EV-associated RNAs have been found in diverse fungi and were proposed to be relevant for pathogenesis in animal hosts. In plant-pathogen interactions, small RNAs are exchanged in a cross-kingdom RNAi warfare and [...] Read more.

Extracellular vesicles (EVs) can transfer diverse RNA cargo for intercellular communication. EV-associated RNAs have been found in diverse fungi and were proposed to be relevant for pathogenesis in animal hosts. In plant-pathogen interactions, small RNAs are exchanged in a cross-kingdom RNAi warfare and EVs were considered to be a delivery mechanism. To extend the search for EV-associated molecules involved in plant-pathogen communication, we have characterised the repertoire of EV-associated mRNAs secreted by the maize smut pathogen, Ustilago maydis. For this initial survey, we examined EV-enriched fractions from axenic filamentous cultures that mimic infectious hyphae. EV-associated RNAs were resistant to degradation by RNases and the presence of intact mRNAs was evident. The set of mRNAs enriched inside EVs relative to the fungal cells are functionally distinct from those that are depleted from EVs. mRNAs encoding metabolic enzymes are particularly enriched. Intriguingly, mRNAs of some known effectors and other proteins linked to virulence were also found in EVs. Furthermore, several mRNAs enriched in EVs are also upregulated during infection, suggesting that EV-associated mRNAs may participate in plant-pathogen interactions. Full article

(This article belongs to the Special Issue Signal Transductions in Fungi)

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17 pages, 8503 KiB

Open AccessEditor’s ChoiceArticle

Development of a Simple and Robust Whole Blood Assay with Dual Co-Stimulation to Quantify the Release of T-Cellular Signature Cytokines in Response to Aspergillus fumigatus Antigens

by Chris D. Lauruschkat, Lukas Page, P. Lewis White, Sonja Etter, Helen E. Davies, Jamie Duckers, Frank Ebel, Elisabeth Schnack, Matthijs Backx, Mariola Dragan, Nicolas Schlegel, Olaf Kniemeyer, Axel A. Brakhage, Hermann Einsele, Juergen Loeffler and Sebastian Wurster

J. Fungi 2021, 7(6), 462; https://doi.org/10.3390/jof7060462 - 8 Jun 2021

Cited by 13 | Viewed by 4754

Abstract

Deeper understanding of mold-induced cytokine signatures could promote advances in the diagnosis and treatment of invasive mycoses and mold-associated hypersensitivity syndromes. Currently, most T-cellular immunoassays in medical mycology require the isolation of mononuclear cells and have limited robustness and practicability, hampering their broader [...] Read more.

Deeper understanding of mold-induced cytokine signatures could promote advances in the diagnosis and treatment of invasive mycoses and mold-associated hypersensitivity syndromes. Currently, most T-cellular immunoassays in medical mycology require the isolation of mononuclear cells and have limited robustness and practicability, hampering their broader applicability in clinical practice. Therefore, we developed a simple, cost-efficient whole blood (WB) assay with dual α-CD28 and α-CD49d co-stimulation to quantify cytokine secretion in response to Aspergillus fumigatus antigens. Dual co-stimulation strongly enhanced A. fumigatus-induced release of T-cellular signature cytokines detectable by enzyme-linked immunosorbent assay (ELISA) or a multiplex cytokine assay. Furthermore, T-cell-dependent activation and cytokine response of innate immune cells was captured by the assay. The protocol consistently showed little technical variation and high robustness to pre-analytic delays of up to 8 h. Stimulation with an A. fumigatus lysate elicited at least 7-fold greater median concentrations of key T-helper cell signature cytokines, including IL-17 and the type 2 T-helper cell cytokines IL-4 and IL-5 in WB samples from patients with Aspergillus-associated lung pathologies versus patients with non-mold-related lung diseases, suggesting high discriminatory power of the assay. These results position WB-ELISA with dual co-stimulation as a simple, accurate, and robust immunoassay for translational applications, encouraging further evaluation as a platform to monitor host immunity to opportunistic pathogens. Full article

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31 pages, 6423 KiB

Open AccessEditor’s ChoiceArticle

A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material

by Ander Peña, Rashid Babiker, Delphine Chaduli, Anna Lipzen, Mei Wang, Mansi Chovatia, Jorge Rencoret, Gisela Marques, María Isabel Sánchez-Ruiz, Teeratas Kijpornyongpan, Davinia Salvachúa, Susana Camarero, Vivian Ng, Ana Gutiérrez, Igor V. Grigoriev, Marie-Noëlle Rosso, Angel T. Martínez and Francisco J. Ruiz-Dueñas

J. Fungi 2021, 7(6), 426; https://doi.org/10.3390/jof7060426 - 28 May 2021

Cited by 17 | Viewed by 6157

Abstract

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on [...] Read more.

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H₂O₂-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes. Full article

(This article belongs to the Special Issue Exploiting Fungal Solutions for Today's Challenges)

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24 pages, 9895 KiB

Open AccessEditor’s ChoiceArticle

Stress-Activated Protein Kinase Signalling Regulates Mycoparasitic Hyphal-Hyphal Interactions in Trichoderma atroviride

by Dubraska Moreno-Ruiz, Linda Salzmann, Mark D. Fricker, Susanne Zeilinger and Alexander Lichius

J. Fungi 2021, 7(5), 365; https://doi.org/10.3390/jof7050365 - 6 May 2021

Cited by 17 | Viewed by 5096

Abstract

Trichoderma atroviride is a mycoparasitic fungus used as biological control agent against fungal plant pathogens. The recognition and appropriate morphogenetic responses to prey-derived signals are essential for successful mycoparasitism. We established microcolony confrontation assays using T. atroviride strains expressing cell division cycle 42 [...] Read more.

Trichoderma atroviride is a mycoparasitic fungus used as biological control agent against fungal plant pathogens. The recognition and appropriate morphogenetic responses to prey-derived signals are essential for successful mycoparasitism. We established microcolony confrontation assays using T. atroviride strains expressing cell division cycle 42 (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1) interactive binding (CRIB) reporters to analyse morphogenetic changes and the dynamic displacement of localized GTPase activity during polarized tip growth. Microscopic analyses showed that Trichoderma experiences significant polarity stress when approaching its fungal preys. The perception of prey-derived signals is integrated via the guanosine triphosphatase (GTPase) and mitogen-activated protein kinase (MAPK) signalling network, and deletion of the MAP kinases Trichoderma MAPK 1 (Tmk1) and Tmk3 affected T. atroviride tip polarization, chemotropic growth, and contact-induced morphogenesis so severely that the establishment of mycoparasitism was highly inefficient to impossible. The responses varied depending on the prey species and the interaction stage, reflecting the high selectivity of the signalling process. Our data suggest that Tmk3 affects the polarity-stress adaptation process especially during the pre-contact phase, whereas Tmk1 regulates contact-induced morphogenesis at the early-contact phase. Neither Tmk1 nor Tmk3 loss-of-function could be fully compensated within the GTPase/MAPK signalling network underscoring the crucial importance of a sensitive polarized tip growth apparatus for successful mycoparasitism. Full article

(This article belongs to the Special Issue Signal Transductions in Fungi)

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