Pub Date : 2025-03-04DOI: 10.1016/j.fgb.2025.103973
Thomas Svoboda , Dominik Niederdöckl-Loibl , Andreas Schüller , Karin Hummel , Sarah Schlosser , Ebrahim Razzazi-Fazeli , Joseph Strauss
Proximity labelling that uses promiscuous biotin ligases (BirA) fused to a bait protein is a powerful tool to identify protein interaction partners in vivo under different metabolic or developmental conditions. BirA can also be used to determine protein composition and interaction partners at specific chromatin locations when it is fused with enzymatically-disabled Cas9 (dCas9) and then guided to the location of interest by sgRNAs. We adapted this method (called CasID) for fungal cells using the nitrate assimilation gene cluster of A. nidulans as a model locus and estrogen-inducible expression of the dCas9-BirA fusion to improve condition-specific labelling. For method establishment, we first verified the presence of dCas-BirA and a known transcription factor at the nitrate locus by chromatin immunoprecipitation (ChIP). Results show that both dCas-BirA and the AreA transcription factor are present at the locus of interest under the conditions used for biotinylation. We then optimized the CasID procedure for efficient labelling and background reduction using the CasID-sgRNA strain and two control strains, one lacking the sgRNA and another one lacking the whole CasID system. Here we provide proof-of-concept for the suitability of the method by showing that biotinylated proteins are enriched in the CasID strains in comparison to the controls. After background reduction, 32 proteins remained in two independent experiments exclusively enriched in the Cas-ID-sgRNA strain. Among these proteins was NmrA, an AreA-interacting regulator, and we also found several chromatin-associated proteins. Overall, our results demonstrate that CasID is suitable for locus-specific labelling and identification of chromatin-associated proteins and transcription factors in A. nidulans. However, the high background of proteins that are biotinylated out of chromatin context or unspecifically attach to the affinity purification matrix needs to be addressed by implementing a set of rigorous controls. In summary, we herewith provide a detailed protocol for application of the method that proved to be useful for the identification of novel chromatin-associated proteins and their interaction partners at a specific genomic locus in divers metabolic and developmental conditions.
Author summary
This study demonstrates that locus-specific proteomics can be carried out by dCas-BirA guided proximity labelling in Aspergillus nidulans. For establishment, we targeted the well-described bidirectional promoter region between niaD, a nitrate reductase, and niiA, a nitrite reductase. At this locus we could test by chromatin immunoprecipitation (ChIP) in combination with qPCR if both, the dCas9-BirA fusion as well as a central transcription factor are at the locus under the conditions of our CasID experiment. After this first control step, we considered that unspecific labelling by dCas-BirA during the t
{"title":"Locus-specific chromatin proteomics using dCas-guided proximity labelling in Aspergillus nidulans","authors":"Thomas Svoboda , Dominik Niederdöckl-Loibl , Andreas Schüller , Karin Hummel , Sarah Schlosser , Ebrahim Razzazi-Fazeli , Joseph Strauss","doi":"10.1016/j.fgb.2025.103973","DOIUrl":"10.1016/j.fgb.2025.103973","url":null,"abstract":"<div><div>Proximity labelling that uses promiscuous biotin ligases (BirA) fused to a bait protein is a powerful tool to identify protein interaction partners <em>in vivo</em> under different metabolic or developmental conditions. BirA can also be used to determine protein composition and interaction partners at specific chromatin locations when it is fused with enzymatically-disabled Cas9 (dCas9) and then guided to the location of interest by sgRNAs. We adapted this method (called CasID) for fungal cells using the nitrate assimilation gene cluster of <em>A. nidulans</em> as a model locus and estrogen-inducible expression of the dCas9-BirA fusion to improve condition-specific labelling. For method establishment, we first verified the presence of dCas-BirA and a known transcription factor at the nitrate locus by chromatin immunoprecipitation (ChIP). Results show that both dCas-BirA and the AreA transcription factor are present at the locus of interest under the conditions used for biotinylation. We then optimized the CasID procedure for efficient labelling and background reduction using the CasID-sgRNA strain and two control strains, one lacking the sgRNA and another one lacking the whole CasID system. Here we provide proof-of-concept for the suitability of the method by showing that biotinylated proteins are enriched in the CasID strains in comparison to the controls. After background reduction, 32 proteins remained in two independent experiments exclusively enriched in the Cas-ID-sgRNA strain. Among these proteins was NmrA, an AreA-interacting regulator, and we also found several chromatin-associated proteins. Overall, our results demonstrate that CasID is suitable for locus-specific labelling and identification of chromatin-associated proteins and transcription factors in <em>A. nidulans</em>. However, the high background of proteins that are biotinylated out of chromatin context or unspecifically attach to the affinity purification matrix needs to be addressed by implementing a set of rigorous controls. In summary, we herewith provide a detailed protocol for application of the method that proved to be useful for the identification of novel chromatin-associated proteins and their interaction partners at a specific genomic locus in divers metabolic and developmental conditions.</div></div><div><h3>Author summary</h3><div>This study demonstrates that locus-specific proteomics can be carried out by dCas-BirA guided proximity labelling in <em>Aspergillus nidulans.</em> For establishment, we targeted the well-described bidirectional promoter region between <em>niaD</em>, a nitrate reductase, and <em>niiA</em>, a nitrite reductase. At this locus we could test by chromatin immunoprecipitation (ChIP) in combination with qPCR if both, the dCas9-BirA fusion as well as a central transcription factor are at the locus under the conditions of our CasID experiment. After this first control step, we considered that unspecific labelling by dCas-BirA during the t","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"178 ","pages":"Article 103973"},"PeriodicalIF":2.4,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143574708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-04DOI: 10.1016/j.fgb.2025.103975
Piotr J. Pietras , Monika Chaszczewska-Markowska , Daniel Ghete , Agata Tyczewska , Kamilla Bąkowska-Żywicka
Environmental conditions have a huge impact on the development of all living things but are especially important in the case of single-celled organisms such as Saccharomyces cerevisiae that must respond quickly and appropriately to any change. Many molecular mechanisms of response to stress have been identified in yeast, but only a few reports address physiological and morphological changes. To investigate S. cerevisiae recovery from ten mild stress conditions and to describe the viability and fitness, we performed a series of growth analysis experiments. Moreover, label-free live cell imaging of yeast subjected to ten environmental stresses has been achieved using holotomography - a leading-edge high resolution 3D quantitative phase imaging. We determined that recovery times of yeast cultures subjected to hyperosmotic and sugar starvation stresses were the shortest, as were the doubling times. Substantially lower proliferation capacity was recorded in yeast after applying sugar- and AA starvation, and high pH stresses, compared to control. Furthermore, the stationary growth was much shorter after subjecting yeast to hypoosmotic and heat stresses, and much longer after anaerobic and UV stresses. Further, we determined changes in shape, colony formation, cell wall damage, volume, sphericity, protein and lipid contents in yeast cells under stress conditions. The most prominent changes were observed for UV and hyperosmotic stresses. Condluding, stress conditions applied to yest cultures affected them differently, causing detrimental effects to their growth, metabolism, fitness and morphology. Moreover, we have proven that holotomography is excellent for precisely determining morphological changes of single cells.
{"title":"Saccharomyces cerevisiae recovery from various mild abiotic stresses: Viability, fitness, and high resolution three-dimensional morphology imaging","authors":"Piotr J. Pietras , Monika Chaszczewska-Markowska , Daniel Ghete , Agata Tyczewska , Kamilla Bąkowska-Żywicka","doi":"10.1016/j.fgb.2025.103975","DOIUrl":"10.1016/j.fgb.2025.103975","url":null,"abstract":"<div><div>Environmental conditions have a huge impact on the development of all living things but are especially important in the case of single-celled organisms such as <em>Saccharomyces cerevisiae</em> that must respond quickly and appropriately to any change. Many molecular mechanisms of response to stress have been identified in yeast, but only a few reports address physiological and morphological changes. To investigate <em>S. cerevisiae</em> recovery from ten mild stress conditions and to describe the viability and fitness, we performed a series of growth analysis experiments. Moreover, label-free live cell imaging of yeast subjected to ten environmental stresses has been achieved using holotomography - a leading-edge high resolution 3D quantitative phase imaging. We determined that recovery times of yeast cultures subjected to hyperosmotic and sugar starvation stresses were the shortest, as were the doubling times. Substantially lower proliferation capacity was recorded in yeast after applying sugar- and AA starvation, and high pH stresses, compared to control. Furthermore, the stationary growth was much shorter after subjecting yeast to hypoosmotic and heat stresses, and much longer after anaerobic and UV stresses. Further, we determined changes in shape, colony formation, cell wall damage, volume, sphericity, protein and lipid contents in yeast cells under stress conditions. The most prominent changes were observed for UV and hyperosmotic stresses. Condluding, stress conditions applied to yest cultures affected them differently, causing detrimental effects to their growth, metabolism, fitness and morphology. Moreover, we have proven that holotomography is excellent for precisely determining morphological changes of single cells.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"178 ","pages":"Article 103975"},"PeriodicalIF":2.4,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143574710","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-01DOI: 10.1016/j.fgb.2025.103971
Jieying Zhu , Weiwei Zhang , Yani Fan , Wei Deng , Liao Zhang , Shunxian Wang , Xingzhong Liu , Meichun Xiang
Septins, a conserved family of cytoskeletal proteins with GTP-binding domains, play key roles in cell polarity, morphogenesis, cytoskeleton organization, and membrane remodeling. The nematode-trapping fungus Arthrobotrys oligospora can capture and kill nematodes using adhesive networks. It has been highlighted the importance of cell polarity, actin organization, and membrane remodeling in the process of trap formation, but the role of septins in adhesive-network forming remains unclear. In this study, we investigated the functions of AoCDC11, an ortholog of Saccharomyces cerevisiae CDC11, through gene disruption and multiphenotypic analysis. Disruption of AoCDC11 led to reduced trap production and abnormal trap morphology. Compared to the wild type, ΔAoCDC11 mutants significantly reduced trap formation to emerge more vegetative hyphae and produced more incompletely fused adhesive networks (45 % vs. 10 %) by fewer trap loops and septa. Additionally, ΔAoCDC11 mutants exhibited a 36 % reduction in hyphal growth and 88 % decrease in conidiation compared to the wild type. Transcriptomic analysis revealed that AoCDC11 regulated genes involved in trap development, including those related to the cell cycle, anatomical structure development, cellular morphogenesis, vesicle transport, and membrane trafficking. These findings suggest that AoCDC11 plays a crucial role in trap morphogenesis, vegetative growth, and conidiation by modulating multiple biological processes. This study expands our understanding of the functions of septins in morphogenesis and survival strategy of nematode-trapping fungi.
septin是一个具有gtp结合结构域的保守的细胞骨架蛋白家族，在细胞极性、形态发生、细胞骨架组织和膜重塑中起着关键作用。捕获线虫的真菌寡孢节肢菌（Arthrobotrys oligospora）利用黏附网捕获并杀死线虫。人们已经强调了细胞极性、肌动蛋白组织和膜重塑在陷阱形成过程中的重要性，但septin在粘连网络形成中的作用仍不清楚。在这项研究中，我们通过基因破坏和多表型分析来研究AoCDC11的功能，AoCDC11是酿酒酵母CDC11的同源物。AoCDC11的破坏导致圈闭产量减少和圈闭形态异常。与野生型相比，ΔAoCDC11突变体显著减少了诱捕器的形成，以出现更多的营养菌丝，并通过减少诱捕器环和间隔产生更多不完全融合的粘附网络（45% vs. 10%）。此外，ΔAoCDC11突变体与野生型相比，菌丝生长减少了36%，分生率减少了88%。转录组学分析显示，AoCDC11调控了参与陷阱发育的基因，包括与细胞周期、解剖结构发育、细胞形态发生、囊泡运输和膜运输相关的基因。这些发现表明，AoCDC11通过调节多种生物过程，在陷阱形态发生、营养生长和条件化中起着至关重要的作用。本研究扩大了我们对septin在线虫诱捕真菌形态发生和生存策略中的作用的认识。
{"title":"Septin AoCDC11 is involved in trap morphogenesis, conidiation, and vegetative growth in carnivorous Arthrobotrys oligospora","authors":"Jieying Zhu , Weiwei Zhang , Yani Fan , Wei Deng , Liao Zhang , Shunxian Wang , Xingzhong Liu , Meichun Xiang","doi":"10.1016/j.fgb.2025.103971","DOIUrl":"10.1016/j.fgb.2025.103971","url":null,"abstract":"<div><div>Septins, a conserved family of cytoskeletal proteins with GTP-binding domains, play key roles in cell polarity, morphogenesis, cytoskeleton organization, and membrane remodeling. The nematode-trapping fungus <em>Arthrobotrys oligospora</em> can capture and kill nematodes using adhesive networks. It has been highlighted the importance of cell polarity, actin organization, and membrane remodeling in the process of trap formation, but the role of septins in adhesive-network forming remains unclear. In this study, we investigated the functions of <em>AoCDC11</em>, an ortholog of <em>Saccharomyces cerevisiae CDC11</em>, through gene disruption and multiphenotypic analysis. Disruption of <em>AoCDC11</em> led to reduced trap production and abnormal trap morphology. Compared to the wild type, <em>ΔAoCDC11</em> mutants significantly reduced trap formation to emerge more vegetative hyphae and produced more incompletely fused adhesive networks (45 % vs. 10 %) by fewer trap loops and septa. Additionally, <em>ΔAoCDC11</em> mutants exhibited a 36 % reduction in hyphal growth and 88 % decrease in conidiation compared to the wild type. Transcriptomic analysis revealed that AoCDC11 regulated genes involved in trap development, including those related to the cell cycle, anatomical structure development, cellular morphogenesis, vesicle transport, and membrane trafficking. These findings suggest that AoCDC11 plays a crucial role in trap morphogenesis, vegetative growth, and conidiation by modulating multiple biological processes. This study expands our understanding of the functions of septins in morphogenesis and survival strategy of nematode-trapping fungi.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"177 ","pages":"Article 103971"},"PeriodicalIF":2.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143534417","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Galactose-containing polysaccharides in the cell walls of filamentous fungi are vital for hyphal formation, mycelial aggregation, and adhesion. Uridine diphosphate (UDP)-glucose 4-epimerase, an enzyme capable of reversibly converting UDP-glucose to UDP-galactose, plays a key role in galactose metabolism. This study investigates the functional specialization and overlapping roles of UDP-glucose 4-epimerases, UgeA and UgeB, in Aspergillus nidulans. Enzyme activity assays revealed that UgeA catalyzes the interconversion of UDP-glucose and UDP-galactose, while UgeB facilitates both UDP-glucose/UDP-galactose and UDP-N-acetylglucosamine/UDP-N-acetylgalactosamine interconversions. Both UgeA and UgeB successfully restored growth in a yeast gal10 disruptant, indicating their involvement in galactose metabolism in vivo. Additionally, the ugeB disruptant of A. nidulans exhibited growth retardation during galactose metabolism, a defect that was alleviated by complementation with ugeB or multiple-copy expression of ugeA. These findings elucidate the complex interplay between sugar metabolism and cell wall synthesis in filamentous fungi and offer insights for the development of novel antifungal therapies.
丝状真菌细胞壁中含半乳糖的多糖对菌丝的形成、菌丝的聚集和粘附至关重要。尿苷二磷酸(UDP)-葡萄糖4-甲酰基酶是一种能够将UDP-葡萄糖可逆转化为UDP-半乳糖的酶，在半乳糖代谢中起着关键作用。本研究探讨了UDP-glucose 4- epimease UgeA和UgeB在灰曲霉中的功能特化和重叠作用。酶活性分析表明，UgeA可以催化udp -葡萄糖和udp -半乳糖的相互转化，而UgeB可以促进udp -葡萄糖/ udp -半乳糖和udp - n -乙酰氨基葡萄糖/ udp - n -乙酰氨基半乳糖的相互转化。UgeA和UgeB都成功地恢复了酵母半乳糖干扰物的生长，表明它们参与了体内半乳糖代谢。此外，夹竹桃的ugeB干扰物在半乳糖代谢过程中表现出生长迟缓，这一缺陷可以通过与ugeB互补或ugeA的多拷贝表达来缓解。这些发现阐明了丝状真菌糖代谢和细胞壁合成之间的复杂相互作用，并为开发新的抗真菌疗法提供了见解。
{"title":"Functional redundancy and divergence of UDP-glucose 4-epimerases in galactose metabolism and cell wall biosynthesis in Aspergillus nidulans","authors":"Chihiro Kadooka , Shun Yakabe , Daisuke Hira , Taiki Futagami , Masatoshi Goto , Takuji Oka","doi":"10.1016/j.fgb.2025.103972","DOIUrl":"10.1016/j.fgb.2025.103972","url":null,"abstract":"<div><div>Galactose-containing polysaccharides in the cell walls of filamentous fungi are vital for hyphal formation, mycelial aggregation, and adhesion. Uridine diphosphate (UDP)-glucose 4-epimerase, an enzyme capable of reversibly converting UDP-glucose to UDP-galactose, plays a key role in galactose metabolism. This study investigates the functional specialization and overlapping roles of UDP-glucose 4-epimerases, UgeA and UgeB, in <em>Aspergillus nidulans</em>. Enzyme activity assays revealed that UgeA catalyzes the interconversion of UDP-glucose and UDP-galactose, while UgeB facilitates both UDP-glucose/UDP-galactose and UDP-<em>N</em>-acetylglucosamine/UDP-<em>N</em>-acetylgalactosamine interconversions. Both UgeA and UgeB successfully restored growth in a yeast <em>gal10</em> disruptant, indicating their involvement in galactose metabolism <em>in vivo</em>. Additionally, the <em>ugeB</em> disruptant of <em>A. nidulans</em> exhibited growth retardation during galactose metabolism, a defect that was alleviated by complementation with <em>ugeB</em> or multiple-copy expression of <em>ugeA</em>. These findings elucidate the complex interplay between sugar metabolism and cell wall synthesis in filamentous fungi and offer insights for the development of novel antifungal therapies.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"177 ","pages":"Article 103972"},"PeriodicalIF":2.4,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143484618","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-12DOI: 10.1016/j.fgb.2025.103970
Alida van Dijk , Andi M. Wilson , Bianke Marx , Bianca Hough , Benedicta Swalarsk-Parry , Lieschen De Vos , Michael J. Wingfield , Brenda D. Wingfield , Emma T. Steenkamp
Fusarium circinatum, the causal agent of pine pitch canker, is one of the most destructive pathogens of Pinus species worldwide. Infections by this pathogen result in serious mortality of seedlings due to root and root collar disease, and growth reduction in trees due to canker formation and dieback. Although much is known about the population biology, genetics, and genomics of F. circinatum, relatively little is known regarding the molecular basis of pathogenicity in F. circinatum. In this study, a protoplast-based transformation using CRISPR-Cas9-mediated genome editing was utilized to functionally characterize a putative pathogenicity gene in three different strains of the fungus. In silico analyses suggested the gene likely encodes a small secreted protein, and all isolates in which it was deleted displayed significantly reduced vegetative growth and asexual spore production compared to the wild-type isolates. In pathogenicity tests, lesions induced by the deletion mutants on detached Pinus patula branches were significantly shorter than those produced by the wild-types. The putative pathogenicity gene was named Pgs reflecting its role in pathogenicity, growth, and sporulation. Future research will seek to explore the molecular mechanisms underlying the mutant phenotypes observed. Overall, this study represents a significant advance in F. circinatum research as the development and application of a Cas9-mediated gene deletion process opens new avenues for functional gene characterization underlying many of the pathogen's biological traits.
{"title":"CRISPR-Cas9 genome editing reveals that the Pgs gene of Fusarium circinatum is involved in pathogenicity, growth and sporulation","authors":"Alida van Dijk , Andi M. Wilson , Bianke Marx , Bianca Hough , Benedicta Swalarsk-Parry , Lieschen De Vos , Michael J. Wingfield , Brenda D. Wingfield , Emma T. Steenkamp","doi":"10.1016/j.fgb.2025.103970","DOIUrl":"10.1016/j.fgb.2025.103970","url":null,"abstract":"<div><div><em>Fusarium circinatum,</em> the causal agent of pine pitch canker, is one of the most destructive pathogens of <em>Pinus</em> species worldwide. Infections by this pathogen result in serious mortality of seedlings due to root and root collar disease, and growth reduction in trees due to canker formation and dieback. Although much is known about the population biology, genetics, and genomics of <em>F. circinatum</em>, relatively little is known regarding the molecular basis of pathogenicity in <em>F. circinatum.</em> In this study, a protoplast-based transformation using CRISPR-Cas9-mediated genome editing was utilized to functionally characterize a putative pathogenicity gene in three different strains of the fungus. <em>In silico</em> analyses suggested the gene likely encodes a small secreted protein, and all isolates in which it was deleted displayed significantly reduced vegetative growth and asexual spore production compared to the wild-type isolates. In pathogenicity tests, lesions induced by the deletion mutants on detached <em>Pinus patula</em> branches were significantly shorter than those produced by the wild-types. The putative pathogenicity gene was named <em>Pgs</em> reflecting its role in <u><strong>p</strong></u>athogenicity, <u><strong>g</strong></u>rowth, and <u><strong>s</strong></u>porulation. Future research will seek to explore the molecular mechanisms underlying the mutant phenotypes observed. Overall, this study represents a significant advance in <em>F. circinatum</em> research as the development and application of a Cas9-mediated gene deletion process opens new avenues for functional gene characterization underlying many of the pathogen's biological traits.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"177 ","pages":"Article 103970"},"PeriodicalIF":2.4,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143426204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.fgb.2024.103958
Lazarina V. Butkovich , Patrick A. Leggieri , Stephen P. Lillington , Tejas A. Navaratna , Candice L. Swift , Nikola G. Malinov , Thea R. Zalunardo , Oliver B. Vining , Anna Lipzen , Mei Wang , Juying Yan , Vivian Ng , Igor V. Grigoriev , Michelle A. O'Malley
Anaerobic gut fungi of the phylum Neocallimastigomycota are microbes proficient in valorizing low-cost but difficult-to-breakdown lignocellulosic plant biomass. Characterization of different fungal life stages and how they contribute to biomass breakdown are critical for biotechnological applications, yet we lack foundational knowledge about the transcriptional, metabolic, and enzyme secretion behavior of different life stages of anaerobic gut fungi: zoospores, germlings, immature thalli, and mature zoosporangia. A Miracloth-based technique was developed to enrich cell pellets with zoospores - the free-swimming, flagellated, young life stage of anaerobic gut fungi. By contrast, fungal mats contained relatively more vegetative, encysted, mature sporangia that form films. Global gene expression profiles were compared from two sample types (zoospore-enriched cell pellets vs. mature mats) harvested from the anaerobic gut fungal strain Neocallimastix californiae G1. Despite cultures being grown on glucose, the fungal zoospore-enriched samples were transcriptionally primed to encounter plant matter substrate, as evidenced by upregulation of catabolic carbohydrate-active enzymes and putative carbohydrate transporters. Furthermore, we report significant differential gene expression for gene annotation groups, including putative secondary metabolites and transcription factors. Understanding global gene expression differences between the fungal zoospore-enriched cells and mature fungi aid in characterizing fungal development, unmasking gene function, and guiding cultivation conditions and engineering targets to promote enzyme secretion.
新马菌门的厌氧肠道真菌是一种精通低成本但难以分解的木质纤维素植物生物量的微生物。表征不同的真菌生命阶段及其对生物量分解的贡献对生物技术应用至关重要，但我们缺乏关于厌氧肠道真菌不同生命阶段的转录、代谢和酶分泌行为的基础知识：游动孢子、萌发、未成熟的菌体和成熟的游动孢子。研究人员开发了一种基于miracloh的技术，用游动孢子（一种自由游动的、鞭毛的、厌氧肠道真菌的年轻生命阶段）来丰富细胞颗粒。相比之下，真菌垫含有相对较多的营养的、成囊的、成熟的孢子囊，孢子囊形成薄膜。研究人员比较了从厌氧肠道真菌菌株Neocallimastix californiae G1中采集的两种样品类型（富含游动孢子的细胞颗粒和成熟的细胞垫）的全局基因表达谱。尽管培养物生长在葡萄糖上，富含游动孢子的真菌样品被转录启动以遇到植物物质底物，正如分解代谢碳水化合物活性酶和假定的碳水化合物转运蛋白上调所证明的那样。此外，我们报告了基因注释组的显著差异基因表达，包括假定的次级代谢物和转录因子。了解真菌游动孢子富集细胞与成熟真菌之间的全局基因表达差异，有助于表征真菌发育，揭示基因功能，指导培养条件和工程靶点促进酶分泌。
{"title":"Separation of life stages within anaerobic fungi (Neocallimastigomycota) highlights differences in global transcription and metabolism","authors":"Lazarina V. Butkovich , Patrick A. Leggieri , Stephen P. Lillington , Tejas A. Navaratna , Candice L. Swift , Nikola G. Malinov , Thea R. Zalunardo , Oliver B. Vining , Anna Lipzen , Mei Wang , Juying Yan , Vivian Ng , Igor V. Grigoriev , Michelle A. O'Malley","doi":"10.1016/j.fgb.2024.103958","DOIUrl":"10.1016/j.fgb.2024.103958","url":null,"abstract":"<div><div>Anaerobic gut fungi of the phylum Neocallimastigomycota are microbes proficient in valorizing low-cost but difficult-to-breakdown lignocellulosic plant biomass. Characterization of different fungal life stages and how they contribute to biomass breakdown are critical for biotechnological applications, yet we lack foundational knowledge about the transcriptional, metabolic, and enzyme secretion behavior of different life stages of anaerobic gut fungi: zoospores, germlings, immature thalli, and mature zoosporangia. A Miracloth-based technique was developed to enrich cell pellets with zoospores - the free-swimming, flagellated, young life stage of anaerobic gut fungi. By contrast, fungal mats contained relatively more vegetative, encysted, mature sporangia that form films. Global gene expression profiles were compared from two sample types (zoospore-enriched cell pellets vs. mature mats) harvested from the anaerobic gut fungal strain <em>Neocallimastix californiae</em> G1. Despite cultures being grown on glucose, the fungal zoospore-enriched samples were transcriptionally primed to encounter plant matter substrate, as evidenced by upregulation of catabolic carbohydrate-active enzymes and putative carbohydrate transporters. Furthermore, we report significant differential gene expression for gene annotation groups, including putative secondary metabolites and transcription factors. Understanding global gene expression differences between the fungal zoospore-enriched cells and mature fungi aid in characterizing fungal development, unmasking gene function, and guiding cultivation conditions and engineering targets to promote enzyme secretion.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"176 ","pages":"Article 103958"},"PeriodicalIF":2.4,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142924122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.fgb.2024.103954
Haochen Miao , Xueyi Chen , Yun Huang , Shenjun Yu , Yang Wang , Xin Huang , Xin Wei
Candida albicans (C. albicans), a common fungal pathogen, is responsible for infections such as oral candidiasis. Given the widespread misuse of antifungal medications and the increasing resistance, it is critical to explore new strategies to eradicate C. albicans. This study investigates ferroptosis, a form of cell death previously underexplored in fungi, focusing on the role of the fungus-specific protein phosphatase Z1 (PPZ1) in regulating the target of rapamycin complex 1 (TORC1) pathway during tert-butyl hydroperoxide (t-BuOOH)-induced ferroptosis. We demonstrated that ferroptosis induced by t-BuOOH promoted the accumulation of iron-dependent lipid peroxides, leading to the death of C. albicans. Furthermore, PPZ1 deletion impairs TORC1 signaling, activates autophagy, increases sensitivity to ferroptosis following t-BuOOH exposure, and reduces resistance to various antifungal drugs. These findings reveal the role of the PPZ1-TORC1 pathway in ferroptosis and provide a theoretical basis for developing ferroptosis as a novel antifungal strategy to eradicate C. albicans. The potential combined application of ferroptosis and antifungal drugs is expected to improve the efficacy of treating fungal infections.
{"title":"PPZ1-TORC1 pathway mediates ferroptosis and antifungal resistance in Candida albicans","authors":"Haochen Miao , Xueyi Chen , Yun Huang , Shenjun Yu , Yang Wang , Xin Huang , Xin Wei","doi":"10.1016/j.fgb.2024.103954","DOIUrl":"10.1016/j.fgb.2024.103954","url":null,"abstract":"<div><div><em>Candida albicans</em> (<em>C. albicans</em>), a common fungal pathogen, is responsible for infections such as oral candidiasis. Given the widespread misuse of antifungal medications and the increasing resistance, it is critical to explore new strategies to eradicate <em>C. albicans</em>. This study investigates ferroptosis, a form of cell death previously underexplored in fungi, focusing on the role of the fungus-specific protein phosphatase Z1 (PPZ1) in regulating the target of rapamycin complex 1 (TORC1) pathway during tert-butyl hydroperoxide (t-BuOOH)-induced ferroptosis. We demonstrated that ferroptosis induced by t-BuOOH promoted the accumulation of iron-dependent lipid peroxides, leading to the death of <em>C. albicans</em>. Furthermore, <em>PPZ1</em> deletion impairs TORC1 signaling, activates autophagy, increases sensitivity to ferroptosis following t-BuOOH exposure, and reduces resistance to various antifungal drugs. These findings reveal the role of the PPZ1-TORC1 pathway in ferroptosis and provide a theoretical basis for developing ferroptosis as a novel antifungal strategy to eradicate <em>C. albicans</em>. The potential combined application of ferroptosis and antifungal drugs is expected to improve the efficacy of treating fungal infections.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"176 ","pages":"Article 103954"},"PeriodicalIF":2.4,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142873561","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.fgb.2024.103942
Sébastien C. Ortiz, Thomas Easter, Clara Valero, Michael J. Bromley, Margherita Bertuzzi
Germination is the fundamental process whereby fungi transition from the dormant and stress resistant spores into actively replicating cells such as hyphae. Germination is essential for fungal colonization of new environments and pathogenesis, yet this differentiation process remains relatively poorly understood. For filamentous fungi, the study of germination has been limited by the lack of high-throughput, temporal, low cost, and easy-to-use methods of quantifying germination. To this end we have developed an image analysis pipeline to automate the quantification of germination from microscopy images. We have optimized this tool for the fungal pathogen Aspergillus fumigatus and demonstrated its potential applications by evaluating different strains, germination inhibitors, and auxotrophic and antifungal resistant mutants. Finally, we have expanded this tool to a variety of filamentous fungi and developed an easy-to-use web app for the fungal research community.
{"title":"A microscopy-based image analysis pipeline for the quantification of germination of filamentous fungi","authors":"Sébastien C. Ortiz, Thomas Easter, Clara Valero, Michael J. Bromley, Margherita Bertuzzi","doi":"10.1016/j.fgb.2024.103942","DOIUrl":"10.1016/j.fgb.2024.103942","url":null,"abstract":"<div><div>Germination is the fundamental process whereby fungi transition from the dormant and stress resistant spores into actively replicating cells such as hyphae. Germination is essential for fungal colonization of new environments and pathogenesis, yet this differentiation process remains relatively poorly understood. For filamentous fungi, the study of germination has been limited by the lack of high-throughput, temporal, low cost, and easy-to-use methods of quantifying germination. To this end we have developed an image analysis pipeline to automate the quantification of germination from microscopy images. We have optimized this tool for the fungal pathogen <em>Aspergillus fumigatus</em> and demonstrated its potential applications by evaluating different strains, germination inhibitors, and auxotrophic and antifungal resistant mutants. Finally, we have expanded this tool to a variety of filamentous fungi and developed an easy-to-use web app for the fungal research community.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"176 ","pages":"Article 103942"},"PeriodicalIF":2.4,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142774936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.fgb.2024.103956
Jack Wess, Yiheng Hu , Sambasivam Periyannan , Ashley Jones, John P. Rathjen
Wheat stripe rust caused by the fungus Puccinia striiformis f. sp. tritici (Pst) is currently the most destructive disease of wheat. The major control methods which include the deployment of resistant wheat cultivars and application of chemical fungicides are losing efficiency as the fungus evolves. Natural antagonists of Pst may be an avenue for alternative and environmentally sustainable control of the disease in the field. Here we describe a novel fungus found growing on Pst pustules. We identified the fungus as a novel isolate of the plant endophyte Penicillium coffeae. We present a high-quality reference genome and a comparative transcriptomic analysis used to investigate how the fungus deploys its genes during growth amongst Pst spores. The gene content of the P. coffeae ANU01 genome is suggestive of a generalist that makes use of diverse substrates. An abundance of genes related to lipid, amino acid and carbohydrate metabolism indicate that P. coffeae ANU01 has evolved the ability to exploit nutrient stores in Pst urediniospores. P. coffeae ANU01 deploys a number of biosynthetic gene clusters during growth on Pst spores, potentially to inhibit urediniospores germination and halt defence responses. A number of genes encoding carbohydrate active enzymes are also highly upregulated, suggesting targeting and degradation of Pst urediniospores structures. Alongside carbohydrates, P. coffeae ANU01 appears to target spore lipids as a nutrient source, secreting several highly upregulated lipases. Our findings broaden the understanding of growth associated with rust spores as an evolutionary strategy and provide insight into the genes potentially required for this process.
{"title":"Global transcriptome changes during growth of a novel Penicillium coffeae isolate on the wheat stripe rust fungus, Puccinia striiformis f. sp. tritici","authors":"Jack Wess, Yiheng Hu , Sambasivam Periyannan , Ashley Jones, John P. Rathjen","doi":"10.1016/j.fgb.2024.103956","DOIUrl":"10.1016/j.fgb.2024.103956","url":null,"abstract":"<div><div>Wheat stripe rust caused by the fungus <em>Puccinia striiformis</em> f. <em>sp. tritici</em> (<em>Pst</em>) is currently the most destructive disease of wheat. The major control methods which include the deployment of resistant wheat cultivars and application of chemical fungicides are losing efficiency as the fungus evolves. Natural antagonists of <em>Pst</em> may be an avenue for alternative and environmentally sustainable control of the disease in the field. Here we describe a novel fungus found growing on <em>Pst</em> pustules. We identified the fungus as a novel isolate of the plant endophyte <em>Penicillium coffeae.</em> We present a high-quality reference genome and a comparative transcriptomic analysis used to investigate how the fungus deploys its genes during growth amongst <em>Pst</em> spores. The gene content of the <em>P. coffeae</em> ANU01 genome is suggestive of a generalist that makes use of diverse substrates. An abundance of genes related to lipid, amino acid and carbohydrate metabolism indicate that <em>P. coffeae</em> ANU01 has evolved the ability to exploit nutrient stores in <em>Pst</em> urediniospores. <em>P. coffeae</em> ANU01 deploys a number of biosynthetic gene clusters during growth on <em>Pst</em> spores, potentially to inhibit urediniospores germination and halt defence responses. A number of genes encoding carbohydrate active enzymes are also highly upregulated, suggesting targeting and degradation of <em>Pst</em> urediniospores structures. Alongside carbohydrates, <em>P. coffeae</em> ANU01 appears to target spore lipids as a nutrient source, secreting several highly upregulated lipases<em>.</em> Our findings broaden the understanding of growth associated with rust spores as an evolutionary strategy and provide insight into the genes potentially required for this process.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"176 ","pages":"Article 103956"},"PeriodicalIF":2.4,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143016783","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.fgb.2024.103957
Padraic G. Heneghan, Letal I. Salzberg, Kenneth H. Wolfe
Zymocin-like killer toxins are anticodon nucleases secreted by some budding yeast species, which kill competitor yeasts by cleaving tRNA molecules. They are encoded by virus-like elements (VLEs), cytosolic linear DNA molecules that are also called killer plasmids. To date, toxins of this type have been found only in budding yeast species (Saccharomycotina). Here, we show that the nuclear genomes of many filamentous fungi (Pezizomycotina) contain small clusters of genes coding for a zymocin-like ribonuclease (γ-toxin), a chitinase (toxin α/β-subunit), and in some cases an immunity protein. The γ-toxins from Fusarium oxysporum and Colletotrichum siamense abolished growth when expressed intracellularly in S. cerevisiae. Phylogenetic analysis of glycoside hydrolase 18 (GH18) domains shows that the chitinase genes in the gene clusters are members of the previously described C-II subgroup of Pezizomycotina chitinases. We propose that the Pezizomycotina gene clusters originated by integration of a yeast-like VLE into the nuclear genome, but this event must have been ancient because (1) phylogenetically, the Pezizomycotina C-II chitinases and the Saccharomycotina VLE-encoded toxin α/β subunit chitinases are sister clades with neither of them nested inside the other, and (2) many of the Pezizomycotina toxin cluster genes contain introns, whereas VLEs do not. One of the toxin gene clusters in Fusarium graminearum is a locus that has previously been shown to be under diversifying selection in North American populations of this plant pathogen. We also show that two genera of agaric mushrooms (Basidiomycota) have acquired toxin gene clusters by horizontal transfers from different Pezizomycotina donors.
{"title":"Zymocin-like killer toxin gene clusters in the nuclear genomes of filamentous fungi","authors":"Padraic G. Heneghan, Letal I. Salzberg, Kenneth H. Wolfe","doi":"10.1016/j.fgb.2024.103957","DOIUrl":"10.1016/j.fgb.2024.103957","url":null,"abstract":"<div><div>Zymocin-like killer toxins are anticodon nucleases secreted by some budding yeast species, which kill competitor yeasts by cleaving tRNA molecules. They are encoded by virus-like elements (VLEs), cytosolic linear DNA molecules that are also called killer plasmids. To date, toxins of this type have been found only in budding yeast species (Saccharomycotina). Here, we show that the nuclear genomes of many filamentous fungi (Pezizomycotina) contain small clusters of genes coding for a zymocin-like ribonuclease (γ-toxin), a chitinase (toxin α/β-subunit), and in some cases an immunity protein. The γ-toxins from <em>Fusarium oxysporum</em> and <em>Colletotrichum siamense</em> abolished growth when expressed intracellularly in <em>S. cerevisiae</em>. Phylogenetic analysis of glycoside hydrolase 18 (GH18) domains shows that the chitinase genes in the gene clusters are members of the previously described C-II subgroup of Pezizomycotina chitinases. We propose that the Pezizomycotina gene clusters originated by integration of a yeast-like VLE into the nuclear genome, but this event must have been ancient because (1) phylogenetically, the Pezizomycotina C-II chitinases and the Saccharomycotina VLE-encoded toxin α/β subunit chitinases are sister clades with neither of them nested inside the other, and (2) many of the Pezizomycotina toxin cluster genes contain introns, whereas VLEs do not. One of the toxin gene clusters in <em>Fusarium graminearum</em> is a locus that has previously been shown to be under diversifying selection in North American populations of this plant pathogen. We also show that two genera of agaric mushrooms (Basidiomycota) have acquired toxin gene clusters by horizontal transfers from different Pezizomycotina donors.</div></div>","PeriodicalId":55135,"journal":{"name":"Fungal Genetics and Biology","volume":"176 ","pages":"Article 103957"},"PeriodicalIF":2.4,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142933326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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