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Background: Puccinia striiformis f. sp. tritici (Pst) causes wheat stripe (yellow) rust disease, which is one of the most destructive diseases affecting wheat worldwide. ATPases, a class of membrane proteins, play an important role in material exchange and signal transduction both within and outside biological cells by transporting ions and phospholipids. In plant pathogens, P-type ATPases primarily participate in pathogen development and virulence regulation. However, the P-type ATPase of subfamily IIIA (PMA) has not yet been identified in Pst. To investigate the potential functions of the PMA gene family in Pst, we conducted a genome-wide bioinformatics analysis and examined the expression profiles of the PMA gene family., Results: Six PMA genes were identified in the genome of P. striiformis f. sp. tritici (CYR34 race). The PMA proteins encoded by these genes ranged in length from 811 to 960 amino acids (aa). Each of the six PMA genes contained a typical ATPase IIIA H superfamily domain and was distributed across four chromosomes. Thirty-six major cis-regulatory elements were detected within the PMA gene family members. Elements such as the CGTCA-motif and TGACG-motif play significant roles in responding to environmental stresses and hormone signals. Quantitative PCR analysis revealed that the expression of the PMA04 gene was generally higher at 9 °C under various temperature stresses. The PMA06 gene typically exhibited higher expression levels at 16 °C. During the infection of Pst, the expression levels of PMA04, PMA05, and PMA06 were elevated at 72 h post treatment., Conclusions: Our results indicate that the PMA gene family in the CYR34 strain comprises six PMA genes, which are crucial for managing temperature stress and pathogen infection, and exhibit a distinctive splicing pattern. This study not only identifies a target and direction for the development of new, efficient, and environmentally friendly control agents for wheat stripe rust but also establishes a foundation for analyzing its pathogenic mechanisms., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. I confirm that I understand BMC Genomics is an open access journal that levies an article processing charge per articles accepted for publication. Competing interests: I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper. The results/data/figures in this manuscript have not been published elsewhere, nor are they under consideration (from you or one of your Contributing Authors) by another publisher. I have read the Nature Portfolio journal policies on author responsibilities and submit this manuscript in accordance with those policies. All of the material is owned by the authors and/or no permissions are required., (© 2025. The Author(s).)

Trichoderma spp. are among the most studied biocontrol agents. While extensive work has been done to understand Trichoderma antagonistic mechanisms, additional research is needed to fully understand how Trichoderma spp. recognize the pathogen-host and the intra-species variability i frequently observed upon interaction with a specific pathogen-host. This study focuses on elucidating the mechanisms underlying observed phenotypic differences among the T. afroharzianum isolates Th19A and Th4 during confrontation with Fusarium virguliforme by investigating differences in their transcriptome at different stages of interaction. In a dual plate assay, Th19A overgrows F. virguliforme, whereas Th4 forms an inhibition zone. Significant differences were observed in the F. virguliforme transcriptome upon interaction with Th19A compared to Th4 and across the different stages of interaction. GO molecular function categories enriched for F. virguliforme genes differed, indicating possible transcriptional plasticity upon interaction with Th19A versus Th4. Significant transcriptome changes were also observed in T. afroharzianum, with several differences in GO-enriched categories between isolates. Several differentially expressed genes-encoding secreted proteins, including CAZymes and CBM1-domain-containing proteins, were up-regulated in Th19A and Th4 upon interaction with the pathogen, even before physical contact, demonstrating possible volatile-mediated recognition of both isolates by F. virguliforme. This study contributes to a better understanding of the interaction between T. afroharzianum and F. virguliforme, which is crucial for developing efficient biological control programs., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2025 Pimentel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Throughout the life cycle of mushrooms, countless spores are released from the fruiting bodies. The spores have significant implications in the food and medicine industries due to pharmacological effects attributed to their bioactive ingredients. Moreover, high concentration of mushroom spores can induce extrinsic allergic reactions in mushroom cultivation workers. Therefore, it is important to study the bioactive ingredients of medicinal mushroom spores and molecular mechanisms of spore formation to develop healthcare products utilizing medicinal mushroom spores and breed sporeless/low- or high-spore-producing strains. This review summarizes the bioactive compounds of mushroom spores, the influence factors and molecular mechanisms of spore formation. Many bioactive compounds extracted from mushroom spores have a wide range of pharmacological activities. Several exogenous factors such as temperature, humidity, light, nutrients, and culture matrix, and endogenous factors such as metabolism-related enzymes activities and expression levels of genes related to sporulation individually or in combination affect the formation, size, and discharge of spores. The future research directions are also discussed for supplying references to analyze the bioactive compounds of spores and the molecular mechanisms of spore formation in mushrooms., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Protein ubiquitination is usually coupled with proteasomal degradation and is crucial in regulating protein quality. The E3 ubiquitin-protein ligase SCF (Skp1-Cullin-F-box) complex directly recognizes the target substrate via interaction between the F-box protein and the substrate. F-box protein is the determinant of substrate specificity. The limited number of identified ubiquitin ligase-substrate pairs is a major bottleneck in the ubiquitination field. Penicillium oxalicum contains many transcription factors, such as BrlA, CreA, XlnR, and Ace1, conserved in filamentous fungi that regulate the fungal development and transcription of (hemi)cellulase genes. Transcription factor Ace1 (also known as SltA) positively correlated with fungal growth and conidiation and negatively correlated with the expression of (hemi)cellulase genes. A ubiquitin ligase-substrate pair, SCFFbx23-Ace1, is identified in P. oxalicum. Most of PoFbx23 is present in free form within the nucleus. A small portion of PoFbx23 associates with Skp1 to form PoFbx23-Skp1 heterodimer or assembles with the three invariable core components (Skp1, Cul1, and Rbx1) of SCF to form the SCFFbx23 complex. Under glucose signal, PoFbx23 absence (Δfbx23) results in decreased transcription levels of the brlA gene which encodes the master regulator for asexual development and six spore pigmentation genes (abrB→abrA→aygB→arpA→arpB→albA) which encode the proteins in the dihydroxynaphthalene-melanin pathway, along with impaired conidiation. Under cellulose signal, transcription levels of (hemi)cellulase genes in the Δfbx23 mutant are significantly upregulated. When PoFbx23 is present, PoAce1 exists as a full-length version and several low-molecular-weight degraded versions. PoAce1 has polyubiquitin modification. Deleting the Pofbx23 gene does not affect Poace1 gene transcription but results in the remarkable accumulation of all versions of the PoAce1 protein. Accumulated PoAce1 protein is a dysfunctional form that no longer binds promoters of the target gene, including the cellulase genes cbh1 and eg1, the hemicellulase gene xyn11A, and the pigmentation-related gene abrB. PoFbx23 acts as a transcriptional coactivator, recognizing and activating PoAce1, allowing the latter to regulate the transcription of target genes with different effects (activating or repressing) under different signals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Spawn aging poses a substantial challenge to the Agaricus bisporus industry. This study focuses on the role of mitochondrial dysfunction in the aging process of A. bisporus spawn. We conducted a comprehensive comparative transcriptome analysis to elucidate the molecular mechanisms underlying A. bisporus spawn aging. A total of 1620 genes with significant expression changes between the normal and aged spawn were identified, including 917 up-regulated genes and 703 down-regulated genes. Our results revealed a notable down-regulation of genes involved in carbohydrate metabolism, mitochondrial energy metabolism, reactive oxygen species (ROS) scavenging, repair mechanisms for oxidative stress-induced damage, fatty acid β-oxidation, and amino acid degradation in aged A. bisporus spawn. Additionally, we observed a decreased expression of genes involved in critical signal transduction pathways associated with mitochondrial function in aged mycelium as well as genes responsible for maintaining mitochondrial stability. The up-regulated genes in aged spawn mainly affect mitochondrial fission and programmed cell death, impacting mitochondrial function. Overall, the present study first provides evidence for the pivotal role of mitochondrial dysfunction in the aging process of A. bisporus spawn and contributes to the development of targeted strategies to enhance mitochondrial function, mitigate spawn aging, and improve the yield and quality of A. bisporus cultivation.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae , the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2 , a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30 . Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler., Competing Interests: CL, EF, TG, NA, BL, RA, ET, JM, UL, SM, AB No competing interests declared, (© 2024, Lichauco, Foss et al.)

Background: Many different stress signaling pathways converge in a common response: slowdown or arrest cell cycle in the G1 phase. The G1/S transition (called Start in budding yeast) is a key checkpoint controlled by positive and negative regulators. Among them, Whi7 and Whi5 are transcriptional repressors of the G1/S transcriptional program, yeast functional homologs of the Retinoblastoma family proteins in mammalian cells. Under standard conditions, Whi7 plays a lesser role than Whi5 in Start inhibition. However, under cell wall stress, Whi7 is induced and plays a more important role in G1/S control. In this work, we investigated the functional hallmarks of Whi7 and Whi5, which determine their strength as Start inhibitors under cell wall stress., Methods: The response of Saccharomyces cerevisiae to Calcofluor White was investigated to characterize the regulation and function of Whi7 and Whi5 under cell wall stress. To control their protein levels, we used dose-dependent β-estradiol-induced expression and auxin-induced degron protein fusions. We also performed Chromatin Immunoprecipitation assays to investigate Whi7 and Whi5 association with Start promoters and scored cell cycle arrest and re-entry using cell microscopy assays., Results: We found that cell wall stress promoted the specific upregulation of the Whi7 Start repressor. First, although cell wall stress increases Whi7 protein levels, this is not the only determinant behind the Whi7 function in promoting G1 arrest. Indeed, artificial induction of Whi5 at the same protein level resulted in a lower G1 block. Second, under cell wall stress, Whi7 was specifically recruited to SBF-target promoters, independent of the increase in its protein levels or cell cycle stage. Finally, we found that Whi7 protein instability further increased during cell wall stress and that Whi7 degradation triggered advanced cell cycle re-entry., Conclusions: Here, we show that cell wall stress signaling specifically enhances Whi7 function as a Start transcriptional repressor. Importantly, we identified new Whi7-specific regulatory mechanisms that do not operate in the Whi5 repressor. Our results indicate that cells may benefit from stress-specific repressors to ensure the stress-induced G1 arrest and that Whi7 rapid degradation may be particularly important to resume cell cycle upon adaptation., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Aspergillus niger is an important industrial filamentous fungus used to produce organic acids and enzymes. A wide dynamic range of promoters, particularly strong promoters, are required for fine-tuning the regulation of gene expression to balance metabolic flux and achieve the high yields of desired products. However, the limited understanding of promoter architectures and activities restricts the efficient transcription regulation of targets in strain engineering in A. niger., Results: In this study, we identified two functional upstream activation sequences (UAS) located upstream of the core promoters of highly expressed genes in A. niger. We constructed and characterized a synthetic promoter library by fusing the efficient UAS elements upstream of the strong constitute PgpdA promoter in A. niger. It demonstrated that the strength of synthetic promoters was fine-tuned with a wide range by tandem assembly of the UAS elements. Notably, the most potent promoter exhibited 5.4-fold higher activity than the strongest PgpdA promoter reported previously, significantly extending the range of strong promoters. Using citric acid production as a case study, we employed the synthetic promoter library to enhance citric acid efflux by regulating the cexA expression in A. niger. It showed a 1.6-2.3-fold increase in citric acid production compared to the parent strain, achieving a maximum titer of 145.3 g/L., Conclusions: This study proved that the synthetic promoter library was a powerful toolkit for precise tuning of transcription in A. niger. It also underscores the potential of promoter engineering for gene regulation in strain improvement of fungal cell factories., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Saccharomyces cerevisiae is a fungus widely used in the food industry and biofuel industry, whereas it is usually exposed to high sugar stress during the fermentation process. Ca 2+ is a key second messenger of the cell, it can regulate cell metabolism. The present study investigated the effect of the Ca 2+ signal on the activity of key enzymes of carbon metabolism and related gene expression in yeast under high sugar fermentation., Results: The expression of genes encoding hexokinase was up-regulated in the high sugar environment, the activity of hexokinase was increased, glucose transmembrane transport capacity was enhanced, the ability of glucose to enter into glycolytic metabolism was increased, and the expression of genes related to pentose phosphate metabolism, glycerol metabolism and trehalose metabolism was up-regulated in the high glucose with Ca 2+ group., Conclusion: Ca 2+ signal regulates the cellular metabolism of glycerol and trehalose and optimizes the allocation of carbon flow by regulating the key enzymes and related gene expression to enhance the resistance of yeast to high sugar stress. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Hyphopichia pseudoburtonii, is emerging as a potential biocontrol agent against various phytopathogens. These traits have been attributed to the production of various antifungal compounds in the presence of target pathogens. However, the broad molecular mechanisms involved in the antifungal activity are not yet understood. This study employed RNA sequencing to assess the temporal changes in H. pseudoburtonii Y963 gene expression patterns when co-cultivated with Botrytis cinerea. Genes differentially expressed in H. pseudoburtonii in co-culture with B. cinerea, compared to the monoculture were evaluated after 24, 48, and 120 h of growth. Up-regulation of genes encoding major core histones (H2A, H3, H4) and ribosomes in the first 24 h suggested an abundance of cells in the S phase of the cell cycle. At 48 h, the genes up-regulated highlight mitotic cell cycle activity and induction of filamentous growth, while in later stages, up-regulation of genes encoding high affinity transporters of sugars, copper and iron, as well as those involved in the retention and utilization of siderophore-iron was evident. Altogether, the data allude to competition for space and nutrients as key mechanisms activated in H. pseudoburtonii in the presence of B. cinerea. This research offers new insights into H. pseudoburtonii transcriptomic response to B. cinerea and illuminates the adaptive strategies and molecular mechanisms behind its antifungal activity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Maluleke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In baker's yeast, genes encoding ribosomal proteins often exist as duplicate pairs, typically with one 'major' paralog highly expressed and a 'minor' less expressed paralog that undergoes controlled expression through reduced splicing efficiency. In this study, we investigate the regulatory mechanisms controlling splicing of the minor paralog of the uS4 protein gene (RPS9A), demonstrating that its splicing is repressed during vegetative growth but upregulated during meiosis. This differential splicing of RPS9A is mediated by two transcription factors, Rim101 and Taf14. Deletion of either RIM101 or TAF14 not only induces the splicing and expression of RPS9A with little effect on the major paralog RPS9B, but also differentially alters the splicing of reporter constructs containing only the RPS9 introns. Both Rim101 and Taf14 co-immunoprecipitate with the chromatin and RNA of the RPS9 genes, indicating that these transcription factors may affect splicing co-transcriptionally. Deletion of the RPS9A intron, RIM101 or TAF14 dysregulates RPS9A expression, impairing the timely expression of RPS9 during meiosis. Complete deletion of RPS9A impairs the expression pattern of meiotic genes and inhibits sporulation in yeast. These findings suggest a regulatory strategy whereby transcription factors modulate the splicing of duplicated ribosomal protein genes to fine-tune their expression in different cellular states., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Filamentous fungi are important producers of enzymes and secondary metabolites. The industrial thermophilic species, Thermothelomyces thermophilus, is closely related to the model fungus, Neurospora crassa . A critical aspect of the filamentous fungal life cycle is the production of asexual spores (conidia), which are regulated by various stimuli, including nutrient availability. Several species of fungi, including T. thermophilus , produce conidia under submerged fermentation conditions, which can be detrimental to product yields. In this study, transcriptional profiling of T. thermophilus was used to map changes during asexual development in submerged cultures, which revealed commonalities of regulation between T. thermophilus and N. crassa . We further identified a transcription factor, res1 , whose deletion resulted in a complete loss of conidia production under fermentation conditions, but which did not affect conidiation on plates. Under fermentation conditions, the ∆ res1 deletion strain showed increased biomass production relative to the wild-type strain, indicating that the manipulation of res1 in T. thermophilus has the potential to increase productivity in industrial settings. Overexpression of res1 caused a severe growth defect and early conidia production on both plates and in submerged cultures, indicating res1 overexpression can bypass regulatory aspects associated with conidiation on plates. Using chromatin-immunoprecipitation sequencing, we identified 35 target genes of Res1, including known conidiation regulators identified in N. crassa , revealing common and divergent aspects of asexual reproduction in these two species.IMPORTANCEFilamentous fungi, such as Thermothelomyces thermophilus, are important industrial species and have been harnessed in the Biotechnology industry for the production of industrially relevant chemicals and proteins. However, under fermentation conditions, some filamentous fungi will undergo a switch from mycelial growth to asexual development. In this study, we use transcriptional profiling of asexual development in T. thermophilus and identify a transcription factor that specifically regulates the developmental switch to the production of unwanted asexual propagules under fermentation conditions, thus altering secreted protein production. Mutations in this transcription factor Res1 result in the loss of asexual development in submerged cultures but do not affect asexual sporulation when exposed to air. The identification of stage-specific developmental regulation of asexual spore production and comparative analyses of conidiation in filamentous ascomycete species have the potential to further manipulate these species for industrial advantage., Competing Interests: The authors declare that the inhibition of conidia production via a ∆res1 mutation in submerged cultures of T. thermophilus has been applied for as a patent (Publication Number WO/2024/107812) by the authors S.H., N.L.G., Y.L., L.B.H., and F.D. This study has been funded by BASF SE without any influence on the design or results of this study.

As the most abundant eukaryotic mRNA modification, N 6 -methyladenosine (m 6 A) plays a crucial role in regulating multiple biological processes. This methylation is regulated by methyltransferases and demethylases. However, the regulatory role and mode of action of m 6 A demethylases in fungi remain poorly understood. In this study, we demonstrate that CpALKBH is a demethylase in Cryphonectria parasitica that removes m 6 A modification from single-stranded RNA in vitro . The deletion of CpALKBH resulted in a significant increase in the m 6 A methylation levels, along with decreases in the growth rate, sporulation, and virulence in C. parasitica . Additionally, CpZap1-a transcription factor-was identified as a downstream target of CpALKBH demethylase based on RNA sequencing analysis. We confirmed that CpALKBH demethylase regulates CpZap1 mRNA stability in an m 6 A-dependent manner. Furthermore, through MazF assay, we found that methylation of CpZap1 at position 1935A is regulated by both CpALKBH demethylase and CpMTA1 methyltransferase. CpZap1 significantly influences the fungal phenotype and virulence, thereby restoring the abnormal phenotype observed in ∆ CpALKBH mutants. Collectively, our findings highlight the essential role of CpALKBH as an m 6 A demethylase in the development and virulence of C. parasitica , while also elucidating the molecular mechanisms through which m 6 A modification impacts CpZap1 mRNA stability., Importance: N 6 -methyladenosine (m 6 A) is the most abundant eukaryotic mRNA modification and is involved in various biological processes. Methyltransferases and demethylases regulate the m 6 A modification, but the regulatory role of m 6 A demethylases in fungi remains poorly understood. Here, we demonstrated that CpALKBH functions as a demethylase in Cryphonectria parasitica . The deletion of CpALKBH leads to a significant increase in m 6 A levels and a reduction in fungal growth, sporulation, and virulence. We identified CpZap1 as a downstream target of CpALKBH, with CpALKBH regulating CpZap1 mRNA stability in an m 6 A-dependent manner. Additionally, our findings indicate that methylation at position 1935A of CpZap1 is regulated by both the CpALKBH demethylase and the CpMTA1 methyltransferase. Given its critical role in fungal development and virulence, overexpression of CpZap1 can rescue abnormal phenotypes of ∆ CpALKBH mutant. Overall, these findings contribute to improving our understanding of the role of m 6 A demethylase in fungi., Competing Interests: The authors declare no conflict of interest.

The fission yeast PHO regulon genes pho1 , pho84 , and tgp1 -encoding a cell surface-associated acid phosphatase (Pho1), a plasma membrane inorganic phosphate transporter (Pho84), and a plasma membrane glycerophosphocholine transporter (Tgp1)-are strongly upregulated in response to acute phosphate starvation, as are the SPBPB2B2.06c and SPAC1039.02 genes that encode putative 5'-nucleotidase paralogs of the binuclear metallophosphoesterase enzyme superfamily. Via proteomic analysis of the medium harvested from phosphate-replete and phosphate-starved fission yeast, we define a starvation secretome that includes SPBPB2B2.06c (renamed Efn1, for e xtracellular f ive-prime n ucleotidase), SPAC1039.02 (henceforth Efn2), and Pho1 among the most abundant exported proteins elaborated by phosphate-starved cells. We demonstrate and characterize a 5'-nucleotidase activity secreted into the medium of phosphate-starved efn1 + efn2 + cells, which is eliminated by simultaneous deletion of efn1 and efn2 . By singly deleting efn1 and efn2 , we find that Efn1 contributes the greater share of secreted 5'-nucleotidase activity. Efn1 and Efn2 catalyze the release of inorganic phosphate from all four standard ribonucleoside monophosphates, in order of preference: CMP > UMP > AMP > GMP. Whereas efn1 + efn2 + cells can use extracellular CMP as a source of phosphate during phosphate starvation, efn1 ∆ efn2 ∆ cells cannot. The secretion of 5'-nucleotidase enzymes during phosphate limitation is a newly appreciated facet of fission yeast phosphate homeostasis., Importance: Schizosaccharomyces pombe adapts to phosphate starvation by upregulating the expression of a cell surface acid phosphatase that mobilizes inorganic phosphate from the extracellular milieu, as well as transmembrane transporters that take up inorganic phosphate and glycerophosphocholine. This study identifies two paralogous extracellular 5'-nucleotidase enzymes, Efn1 and Efn2, encoded by genes that are highly transcriptionally induced during acute phosphate starvation, as major proteins secreted into the medium by phosphate-starved fission yeast cells. Secreted Efn1 and Efn2 catalyze the release of inorganic phosphate from all ribonucleoside monophosphates, with a preference for CMP. Secretion of Efn1 and Efn2 enables phosphate-starved fission yeast to thrive by using extracellular CMP as a source of inorganic phosphate. The starvation-induced production of extracellular 5'-nucleotidases adds a new layer of pro-adaptive function during phosphate limitation., Competing Interests: The authors declare no conflict of interest.

Transcription factors (TFs) involved in sexual reproduction in filamentous fungi have been characterized. However, we have little understanding of how these TFs synergize within regulatory networks resulting in sexual development. We investigated 13 TFs in Fusarium graminearum , whose knockouts exhibited abortive or arrested phenotypes during sexual development to elucidate the transcriptional regulatory cascade underlying the development of the sexual fruiting bodies. A Bayesian network of the TFs was inferred based on transcriptomic data from key stages of sexual development. We evaluated in silico knockout impacts to the networks of the developmental phenotypes among the TFs and guided knockout transcriptomics experiments to properly assess regulatory roles of genes with same developmental phenotypes. Additional transcriptome data were collected for the TF knockouts guided by the stage at which their phenotypes appeared and by the cognate in silico prediction. Global TF networks revealed that TFs within the mating-type locus ( MAT genes) trigger a transcriptional cascade involving TFs that affected early stages of sexual development. Notably, PNA1 , whose knockout mutants produced exceptionally small protoperithecia, was shown to be an upstream activator for MAT genes and several TFs essential for ascospore production. In addition, knockout mutants of SUB1 produced excessive numbers of protoperithecia, wherein MAT genes and pheromone-related genes exhibited dysregulated expression. We conclude that PNA1 and SUB1 play central and suppressive roles in initiating sexual reproduction, respectively. This comprehensive investigation contributes to our understanding of the transcriptional framework governing the multicellular body plan during sexual reproduction in F. graminearum .IMPORTANCEUnderstanding transcriptional regulation of sexual development is crucial to the elucidation of the complex reproductive biology in Fusarium graminearum . We performed gene knockouts on 13 transcription factors (TFs), demonstrating knockout phenotypes affecting distinct stages of sexual development. Using transcriptomic data across stages of sexual development, we inferred a Bayesian network of these TFs that guided experiments to assess the robustness of gene interactions using a systems biology approach. We discovered that the mating-type locus ( MAT genes) initiates a transcriptional cascade, with PNA1 identified as an upstream activator essential for early sexual development and ascospore production. Conversely, SUB1 was found to play a suppressive role, with knockout mutants exhibiting excessive protoperithecia due to abnormally high expression of MAT and pheromone-related genes. These findings highlight the central roles of PNA1 and SUB1 in regulating other gene activity related to sexual reproduction, contributing to a deeper understanding of the mechanisms of the multiple TFs that regulate sexual development., Competing Interests: The authors declare no conflict of interest.

Cryptococcus neoformans is an opportunistic fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth under host-like conditions. To understand how individual environmental signals affect capsule size and gene expression, we grew cells in all combinations of 5 signals implicated in capsule size and systematically measured cell and capsule sizes. We also sampled these cultures over time and performed RNA-seq in quadruplicate, yielding 881 RNA-seq samples. Analysis of the resulting data sets showed that capsule induction in tissue culture medium, typically used to represent host-like conditions, requires the presence of either CO2 or exogenous cyclic AMP. Surprisingly, adding either of these pushes overall gene expression in the opposite direction from tissue culture media alone, even though both are required for capsule development. Another unexpected finding was that rich medium blocks capsule growth completely. Statistical analysis further revealed many genes whose expression is associated with capsule thickness; deletion of one of these significantly reduced capsule size. Beyond illuminating capsule induction, our massive, uniformly collected data set will be a significant resource for the research community., Competing Interests: Conflicts of interest: The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The fission yeast, Schizosaccharomyces pombe, is an excellent eukaryote model organism for studying essential biological processes. Its genome contains ∼1,200 genes essential for cell viability, most of which are evolutionarily conserved. To study these essential genes, resources enabling conditional perturbation of target genes are required. Here, we constructed comprehensive arrayed libraries of plasmids and strains to knock down essential genes in S. pombe using dCas9-mediated CRISPRi. These libraries cover ∼98% of all essential genes in fission yeast. We estimate that in ∼60% of these strains, transcription of a target gene was repressed so efficiently that cell proliferation was significantly inhibited. To demonstrate the usefulness of these libraries, we performed metabolic analyses with knockdown strains and revealed flexible interaction among metabolic pathways. Libraries established in this study enable comprehensive functional analyses of essential genes in S. pombe and will facilitate the understanding of essential biological processes in eukaryotes., (© 2024 Ishikawa et al.)

Translation initiation, which involves numerous protein factors and coordinated control steps, represents the most complicated process during eukaryotic translation. However, the roles of eukaryotic translation initiation factor (eIF) in filamentous fungi are not well clarified. In this study, we investigated the function of eIF2Bα in Aspergillus oryzae, an industrially important filamentous fungus. The ΔeIF2Bα mutants showed slow colony growth and decreased conidia production, suggesting the critical roles of eIF2Bα in the growth and development of A. oryzae. In addition, the loss of eIF2Bα significantly impaired the ability to produce amylase and kojic acid, indicating the involvement of eIF2Bα in the amylase synthesis and secondary metabolite production. Interestingly, the elimination of eIF2Bα improved the tolerance of A. oryzae to diverse adverse stresses, including endoplasmic reticulum stress, oxidative stress, cell wall-perturbing stress, and cell membrane-damaging stress. Overall, our results indicate that eIF2Bα is a crucial regulator of growth, development, stress response, amylase production, and kojic acid synthesis in A. oryzae., Competing Interests: Declarations. Conflict of Interest: No potential conflict of interest was reported by the authors. Ethical Approval: This article does not contain any studies with human participants or animals performed by any of the authors., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The mycotoxigenic fungi, Aspergillus flavus and Fusarium verticillioides , commonly co-colonize maize in the field, yet their direct interactions at the chemical communication level have not been well characterized. Here, we examined if and how the two most infamous mycotoxins produced by these species, aflatoxin and fumonisin, respectively, govern interspecies growth and mycotoxin production. We showed that fumonisin producing strains of F. verticillioides suppressed the growth of A. flavus while non-producers did not. Additionally, while aflatoxin did not inhibit F. verticillioides growth, it did suppress fumonisin production. Fumonisin B 1 concentration levels plummeted when challenged with a high dose of aflatoxin B 1 or with an aflatoxin producing strain. With these findings, expression of the genetic regulators of secondary metabolism was investigated for both fungi. While no strong effect was seen on genes in the aflatoxin biosynthetic gene cluster when exposed to fumonisin B 1 , the fumonisin repressor FvZBD1 , which is adjacent to the cluster, was induced with expression proportionate to concentration when F. verticillioides was challenged with aflatoxin B 1 . We also assessed the expression of the global regulators of fungal secondary metabolism, veA and laeA , and found that their expression is altered in both A. flavus and F. verticillioides when exposed to their competitor's mycotoxin. This work gives insight into the ecological roles of mycotoxins and why these fungi may produce them as weapons in the interspecies battle for resource acquisition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Satterlee, Hawkins, Mitchell, Wei, Lohmar, Glenn and Gold.)

The assembly of repressive heterochromatin in eukaryotic genomes is crucial for silencing lineage-inappropriate genes and repetitive DNA elements. Paradoxically, transcription of repetitive elements within constitutive heterochromatin domains is required for RNA-based mechanisms, such as the RNAi pathway, to target heterochromatin assembly proteins. However, the mechanism by which heterochromatic repeats are transcribed has been unclear. Using fission yeast, we show that the conserved trimeric transcription factor (TF) PhpC NF-Y complex can infiltrate constitutive heterochromatin via its histone-fold domains to transcribe repeat elements. PhpC NF-Y collaborates with a Zn-finger containing TF to bind repeat promoter regions with CCAAT boxes. Mutating either the TFs or the CCAAT binding site disrupts the transcription of heterochromatic repeats. Although repeat elements are transcribed from both strands, PhpC NF-Y -dependent transcripts originate from only one strand. These TF-driven transcripts contain multiple cryptic introns which are required for the generation of small interfering RNAs (siRNAs) via a mechanism involving the spliceosome and RNAi machinery. Our analyses show that siRNA production by this TF-mediated transcription pathway is critical for heterochromatin nucleation at target repeat loci. This study reveals a mechanism by which heterochromatic repeats are transcribed, initiating their own silencing by triggering a primary cascade that produces siRNAs necessary for heterochromatin nucleation., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Aspergillus cristatus is a dominant fungus formed during the "flowering" process of Fuzhuan brick tea. Previous research has established that the sporulation of Aspergillus nidulans, a model organism of filamentous fungi, is regulated by light. However, the sporulation of A. cristatus is dependent on osmotic stress. In a previous study, we used pull-down and mass spectrometry to identify proteins that interacted with AcHog1 in A. cristatus when cultured under different conditions of osmotic stress. In the present study, we analyzed the proteins we identified previously to investigate their functional role. The AA1E3BER4 protein was located downstream of Hog1 in the HOG branch pathway and was identified that was regulated by AcHog1. Furthermore, yeast two-hybrid analysis showed that AA1E3BER4 interacted with AcHog1. In addition, we knocked out and complemented the Acsko1 gene encoding the AA1E3BER4 protein. We found that the number of sexual and asexual spores were downregulated by 3.81- and 4.57-fold, respectively, in the ΔAcsko1 strain. The sensitivity of the ΔAcsko1 strain to sorbitol and sucrose, as regulators of osmotic stress, increased, and the sensitivity to high sucrose was higher than that of sorbitol. Acsko1 also regulated the response of A. cristatus to oxidative stress, Congo red, and SDS (sodium dodecyl sulfate). In addition, the deletion of Acsko1 significantly increased the pigment of the ΔAcsko1 strain. This is the first study to report the role of the sko1 gene in oxidative stress, stress-induced damage to the cell wall, and pigment in Aspergillus cristatus., (© 2024 Wiley‐VCH GmbH.)

Woronin bodies are unique organelles in Pezizomycotina fungi that allow hyphae compartmentalization and prevent cytoplasmatic bleeding after mechanical injury. Several studies have related the peroxisomal protein HEX1, the major component of Woronin bodies with other biological processes such as hyphal growth, osmotic stress tolerance and pathogenicity. Trichoderma spp. are plant-beneficial multipurpose biological control agents, and proteomic and transcriptomic studies have shown that HEX1 and its corresponding gene are overrepresented when grown in the presence of fungal cell walls and plant polymers. To further investigate the involvement of hex1 in Trichoderma biology, we generated hex1 deletion transformants using the wheat endophytic strain T. simmonsii T137 as host. Results confirmed that hex1 gene is involved in the prevention of cytoplasmatic bleeding, and also has a role in fungal growth and biocontrol potential against phytopathogenic fungi and oomycetes. The involvement of hex1 in the fungal response to osmotic and oxidative stresses is conditioned by the type of stress and by the nutrient richness of the culture medium. The hex1 deletion also affected the interaction with wheat, but did not affect the plant protective effect of T137 against water stress. Overall, this study shows the implication of HEX1 in a wide range of biological processes necessary for T. simmonsii to deploy its abilities to be used as an agriculturally beneficial fungus., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Optogenetic systems utilize genetically encoded light-sensitive proteins to control cellular processes such as gene expression and protein localization. Like most synthetic systems, generation of an optogenetic system with desirable properties requires multiple design-test-build cycles. A yeast optogenetic toolkit (yOTK) allows rapid assembly of optogenetic constructs using Modular Cloning, or MoClo. In this protocol, we describe how to assemble, integrate, and test optogenetic systems in the budding yeast Saccharomyces cerevisiae. Generating an optogenetic system requires the user to first define the structure of the final construct and identify all basic parts and vectors required for the construction strategy, including light-sensitive proteins that need to be domesticated into the toolkit. The assembly is then defined following a set of standard rules. Multigene constructs are assembled using a series of one-pot assembly steps with the identified parts and vectors and transformed into yeast. Screening of the transformants allows optogenetic systems with optimal properties to be selected., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Monacolin K is a valuable secondary metabolite produced after a period of fermentation by Monascus purpureus; however, our current understanding of the regulatory mechanisms of its synthesis remains incomplete. This study conducted functional analysis on the key transcription factor, comp54181_c0, that is involved in the synthesis of monacolin K in Monascus. Mutant strains with either knockout or overexpression of comp54181_c0 were constructed using CRISPR/Cas9. A comparison between the knockout and overexpression strains revealed changes in fungal morphology and growth, with a significant increase in the production of Monascus pigments and monacolin K when comp54181_c0 was absent. Real-time fluorescence quantitative PCR analysis revealed that comp54181_c0 significantly influenced the transcription of key genes related to monacolin K biosynthesis in Monascus. In conclusion, our study elucidates the crucial role of comp54181_c0 in Monascus, enriches our understanding of fungal secondary metabolite development and regulation, and provides a foundation for the development and regulation of Monascus and monacolin K production., (© 2024 Wiley‐VCH GmbH.)

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, seriously endangers the healthy growth of apple trees. Mitogen-Activated Protein Kinase (MAPK) signaling pathway is an important pathway to transmit signals stimulated by environmental stress. In this study, we identify and functionally characterize MAPKKK VmMkh1, MAPKK VmMkk1 and MAPK VmSpm1. VmMkh1 and VmMkk1 positively regulate the phosphorylation of VmSpm1. The radial growth rate of the VmSpm1 deletion mutant was reduced by approximately 31 %. There was no significant difference in growth rate between the VmMkh1 and VmMkk1 mutant and the wild-type. VmMkh1 hyphe branches into a curved shape. The VmMkh1, VmMkk1, and VmSpm1 deletion mutant produced fewer conidia than the wild-type strain at 20 days post inoculation. Moreover, the VmMkh1, VmMkk1, and VmSpm1 deletion mutant slows conidial germination. The hyphal growth of VmMkh1, VmMkk1, and VmSpm1 deletion mutants are significantly inhibited on media containing NaCl, KCl, sorbitol (high osmotic stresses). The hyphal growth of VmMkh1, VmMkk1, and VmSpm1 deletion mutants are significantly inhibited on media containing Congo red, CFW, SDS, and Lysing encymes (Cell wall stress agents). A looser distribution of spacers in VmMkh1, VmMkk1, and VmSpm1 deletion mutants compared with the wild-type strain. The size of lesions on apple fruits and branches inoculated with VmSpm1 deletion mutant showed a reduction of approximately 46 % and 43 %, respectively, after 9 dpi. Overall, our findings demonstrate that VmMkh1, VmMkk1, and VmSpm1 are involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and pathogenicity of Valsa mali., Competing Interests: Declaration of competing interest Conflict of Interest The authors declare that no conflict of interest exists., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The majority of Aspergillus fumigatus reproduction occurs asexually, with large numbers of conidiophores producing small hydrophobic conidia dispersed aerially. When healthy hosts inhale conidia, the mucosal cilia and phagocytosis by the innate immune system can remove them. However, in immunocompromised hosts, the conidia are not removed, which allows them to germinate, forming mycelium that invades host tissues and causes disease. Previously we isolated a white A. fumigatus A1j strain incapable of producing conidia and screened several genes (including dopA) with significant expression differences and mutant loci in A1j. DopA homologous proteins in other species have been partially studied and are known to participate in various membrane transport-related cellular functions. Defects in these proteins in Saccharomyces cerevisiae, Caenorhabditis elegans, and Aspergillus nidulans result in defective cell morphology and abnormal growth. In this study, we observed reduced conidia production and abnormal development of spore-producing structures in the A. fumigatus dopA null strain, compared to parental strain, and demonstrated that dopA also modulates stress response and virulence of A. fumigatus., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Transcriptomics is a powerful approach for functional genomics and systems biology, yet it can also be used for genetic part discovery. Here, we derive constitutive and light-regulated promoters directly from transcriptomics data of the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938 (anamorph Phaffia rhodozyma) and use these promoters with other genetic elements to create a modular synthetic biology parts collection for this organism. X. dendrorhous is currently the sole biotechnologically relevant yeast in the Tremellomycete class-it produces large amounts of astaxanthin, especially under oxidative stress and exposure to light. Thus, we performed transcriptomics on X. dendrorhous under different wavelengths of light (red, green, blue, and ultraviolet) and oxidative stress. Differential gene expression analysis (DGE) revealed that terpenoid biosynthesis was primarily upregulated by light through crtI, while oxidative stress upregulated several genes in the pathway. Further gene ontology (GO) analysis revealed a complex survival response to ultraviolet (UV) where X. dendrorhous upregulates aromatic amino acid and tetraterpenoid biosynthesis and downregulates central carbon metabolism and respiration. The DGE data was also used to identify 26 constitutive and regulated genes, and then, putative promoters for each of the 26 genes were derived from the genome. Simultaneously, a modular cloning system for X. dendrorhous was developed, including integration sites, terminators, selection markers, and reporters. Each of the 26 putative promoters were integrated into the genome and characterized by luciferase assay in the dark and under UV light. The putative constitutive promoters were constitutive in the synthetic genetic context, but so were many of the putative regulated promoters. Notably, one putative promoter, derived from a hypothetical gene, showed ninefold activation upon UV exposure. Thus, this study reveals metabolic pathway regulation and develops a genetic parts collection for X. dendrorhous from transcriptomic data. Therefore, this study demonstrates that combining systems biology and synthetic biology into an omics-to-parts workflow can simultaneously provide useful biological insight and genetic tools for nonconventional microbes, particularly those without a related model organism. This approach can enhance current efforts to engineer diverse microbes. KEY POINTS: • Transcriptomics revealed further insights into the photobiology of X. dendrorhous, specifically metabolic nodes that are transcriptionally regulated by light. • A modular genetic part collection was developed, including 26 constitutive and regulated promoters derived from the transcriptomics of X. dendrorhous. • Omics-to-parts can be applied to nonconventional microbes for rapid "onboarding"., Competing Interests: Declarations. Ethics approval: This article does not contain any studies with human participants or animals performed by any of the authors. Disclosure: The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of ODNI, IARPA, or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation therein. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s).)

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce. In-depth analysis of transcriptome data from FLO8-deficient K. phaffii revealed that Flo8 affects genes involved in cell cycle, mating, respiration, and catabolite repression additionally to flocculation targets. One gene with considerably increased expression in flo8 was GTH1, encoding a high-affinity glucose transporter in K. phaffii. Its promoter (P G1 ) was previously established as a strong, glucose-regulatable alternative to methanol-induced promoters. P G1 and its improved derivatives P G1 - 3 , D-P GS4 and D-P GS5 , proved to be promising candidates for controlling recombinant protein production in the FLO8-deficient background. In small-scale screenings, P G1 3 -controlled intracellular EGFP levels were 2.8-fold higher, and yields of different secreted recombinant proteins were up to 4.8-fold increased. The enhanced productivity of the flo8 mutant in combination with the P G1 variants was transferrable to glucose-limited fed-batch processes and could largely be attributed to higher transcriptional activity of the promoter, leading to a much higher productivity per chromosomally integrated gene copy. K. phaffii flo8 has many advantageous characteristics, such as reduced surface growth and increased transcriptional strength of glucose-regulatable promoters. These features turn the flo8 strain into a valuable new base strain for various experimental designs and establish flo8 as an excellent strain background for methanol-free recombinant protein production processes., Competing Interests: Declaration of Competing Interest All authors have read the manuscript and agree with its publication. The authors declare that they have no conflict of interest. WO2015158800A1 patent family covers the use of flo8 for protein production in K. phaffii (inventors B. Gasser, D. Mattanovich, M. Buchetics; application by Lonza Ltd, Boehringer Ingelheim RCV GmbH, Sandoz GmbH and Validogen GmbH). Some of the authors (B. Gasser, C. Rebnegger, D. Mattanovich, M. Flores-Villegas) are inventors, but not owners, of the patent family covering the surprising effect of the flo8 deletion on the P(G)-promoter variants (WO2020144313A1 by Lonza Ltd)., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The filamentous fungus Trichoderma reesei is extensively utilized in the realm of recombinant protein expression owing to its well-established protein production systems. However, the potential for efficient and convenient protein production in T. reesei has not been fully harnessed. To further increase the production of recombinant lipase Candida antarctica lipase B (CalB), we overexpressed seven transcription activators and found that overexpression of the calcineurin CRZ1 could significantly enhance CalB production by 2.08-fold, and then through employing a synthetic strong promoter Pcbh7IR and knocking out ace1 , a competitor of crz1 , CalB production was further increased 4.2-fold. Furthermore, we found that increasing the Ca 2+ concentration enabled the enhancement of CalB production in the CRZ1-overexpressing strains. Additionally, transcription analysis revealed a significant upregulation of calcium-related pathway genes and ER chaperone genes in T. reesei , revealing that overexpression of CRZ1 can increase the production of the recombinant protein by activation of the calcium signaling pathway. In this study, by systematically manipulating the recombinant protein expression module and its regulatory transcription activators, we found a positive effect of crz1 -mediated calcium channels in boosting the expression of the recombinant protein.

The Hog1 stress-activated protein kinase (SAPK) is a key mediator of stress resistance and virulence in Candida albicans. Hog1 activation via phosphorylation of the canonical TGY motif is mediated by the Pbs2 MAPKK, which itself is activated by the Ssk2 MAPKKK. Although this three-tiered SAPK signalling module is well characterised, it is unclear how Hog1 activation is regulated in response to different stresses. Functioning upstream of the Ssk2 MAPKKK is a two-component related signal transduction system comprising three sensor histidine kinases, a phosphotransfer protein Ypd1, and a response regulator Ssk1. Here, we report that Ssk1 is a master regulator of the Hog1 SAPK that promotes stress resistance and Hog1 phosphorylation in response to diverse stresses, except high osmotic stress. Notably, we find Ssk1 regulates Hog1 in a two-component independent manner by functioning to promote interactions between the Ssk2 and Pbs2 kinases. We propose this function of Ssk1 is important to maintain a basal level of Hog1 phosphorylation which is necessary for oxidative stress, but not osmotic stress, mediated Hog1 activation. We find that osmotic stress triggers robust Pbs2 phosphorylation which drives its dissociation from Ssk2. In contrast, Pbs2 is not robustly phosphorylated following oxidative stress and the Ssk1-mediated Ssk2-Pbs2 interaction remains intact. Instead, oxidative stress-stimulated increases in phosphorylated Hog1 is dependent on the inhibition of protein tyrosine phosphatases that negatively regulate Hog1 coupled with the Ssk1-mediated promotion of basal Hog1 activity. Furthermore, we find that inhibition of protein tyrosine phosphatases is linked to the hydrogen peroxide induced oxidation of these negative regulators in a mechanism that is partly dependent on thioredoxin. Taken together these data reveal stress contingent changes in Hog1 pathway architecture and regulation and uncover a novel mode of action of the Ssk1 response regulator in SAPK regulation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Day et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Organization of the eukaryotic genome is essential for proper function, including gene expression. In metazoans, chromatin loops and Topologically Associated Domains (TADs) organize genes into transcription factories, while chromosomes occupy nuclear territories in which silent heterochromatin is compartmentalized at the nuclear periphery and active euchromatin localizes to the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation typified by heterochromatic centromeres and telomeres independently clustering at the nuclear membrane, while interspersed heterochromatic loci aggregate across Megabases of linear genomic distance to loop chromatin in TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown., Results: We examined the genome organization of a Neurospora strain harboring a ~ 47.4 kilobase deletion within a temporarily silent, facultative heterochromatic region, as well as the genome organization of a strain deleted of a 110.6 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion minimally effects local chromatin structure or telomere clustering, the constitutive heterochromatin deletion alters local chromatin structure, the predicted three-dimensional chromosome conformation, and the expression of some genes, which are qualitatively repositioned into the nucleus center, while increasing Hi-C variability., Conclusions: Our work elucidates how an individual constitutive heterochromatic region impacts genome organization and function. Specifically, one silent region indirectly assists in the hierarchical folding of the entire Neurospora genome by aggregating into the "typical" heterochromatin bundle normally observed in wild type nuclei, which may promote normal gene expression by positioning euchromatin in the nucleus center., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Blue light illumination can be detected by light-oxygen-voltage (LOV) photosensing proteins and translated into a range of biochemical responses, facilitating the generation of novel optogenetic tools to control cellular function. Here, we develop new variants of our previously described VP-EL222 light-dependent transcription factor and apply them to study the phosphate-responsive signaling ( PHO ) pathway in the budding yeast Saccharomyces cerevisiae , exemplifying the utilities of these new tools. Focusing first on the VP-EL222 protein itself, we quantified the tunability of gene expression as a function of light intensity and duration and demonstrated that this system can tolerate the addition of substantially larger effector domains without impacting function. We further demonstrated the utility of several EL222-driven transcriptional controllers in both plasmid and genomic settings, using the PHO5 and PHO84 promoters in their native chromosomal contexts as examples. These studies highlight the utility of light-controlled gene activation using EL222 tethered to either artificial transcription domains or yeast activator proteins (Pho4). Similarly, we demonstrate the ability to optogenetically repress gene expression with EL222 fused to the yeast Ume6 protein. We finally investigated the effects of moving EL222 recruitment sites to different locations within the PHO5 and PHO84 promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (P i ) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionalities that they can deliver and the biological questions that can be probed.

Maf1 is a general and global negative regulator of RNA polymerase III (Pol III) transcription. Under repressive conditions, Maf1 binds directly to the Pol III complex and sequesters Pol III elements that are involved in transcription initiation. To further understand Pol III regulation, we searched for genetic bypass suppressors of a maf1 deletion mutant (maf1Δ) of Saccharomyces cerevisiae. Strains that carried maf1Δ were temperature-sensitive on media that contained nonfermentable carbon sources and showed the antisuppressor phenotype. Suppressors allowed colonies to grow at the restrictive temperature on glycerol media and partially complemented the antisuppressor phenotype of maf1Δ. DNA plasmids that were identified as overdose suppressors encoded N-terminal fragments of the largest Pol III subunit, C160 of various lengths. The shortest fragment, 372 amino acids long, the overdose of which partially complemented the antisuppressor phenotype and temperature-sensitive respiratory growth of maf1Δ, was named C160-NTF. In this study, we showed that the expression of HA-tagged C160-NTF resulted in accumulation of approximately 40 kDa protein that was distributed throughout the yeast cell, in the cytoplasm and nucleus. The overdose of C160-NTF led to decrease of tRNA transcription in maf1Δ mutant cells, demonstrating functional suppression. Levels of newly synthesized individual tRNAs and Pol III occupancies on tRNA genes were decreased by C160-NTF in the maf1Δ mutant. Additionally, we analyzed the effect of C160-NTF overproduction and the presence of Maf1 on the associations among Pol III subunits. Previous structural analyzes of Pol III have indicated that the N-terminal region of C160 interacts with the C82-34-31 heterotrimeric Pol III subcomplex. We suggest that the negative effect of C160-NTF overdose on tRNA transcription is related to defective Pol III assembly, because overproduction of C160-NTF altered C160 interactions with C34 and C82 in the maf1Δ mutant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Starvation, which is associated with inactivation of the growth-promoting TOR complex 1 (TORC1), is a strong environmental signal for cell differentiation. In the fission yeast Schizosaccharomyces pombe, nitrogen starvation has distinct physiological consequences depending on the presence of mating partners. In their absence, cells enter quiescence, and TORC1 inactivation prolongs their life. In presence of compatible mates, TORC1 inactivation is essential for sexual differentiation. Gametes engage in paracrine pheromone signaling, grow towards each other, fuse to form the diploid zygote, and form resistant, haploid spore progenies. To understand the signaling changes in the proteome and phospho-proteome during sexual reproduction, we developed cell synchronization strategies and present (phospho-)proteomic data sets that dissect pheromone from starvation signals over the sexual differentiation and cell-cell fusion processes. Unexpectedly, these data sets reveal phosphorylation of ribosomal protein S6 during sexual development, which we establish requires TORC1 activity. We demonstrate that TORC1 is re-activated by pheromone signaling, in a manner that does not require autophagy. Mutants with low TORC1 re-activation exhibit compromised mating and poorly viable spores. Thus, while inactivated to initiate the mating process, TORC1 is reactivated by pheromone signaling in starved cells to support sexual reproduction., Competing Interests: SGM is a member of the PLOS Biology Editorial Board., (Copyright: © 2024 Bérard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The plasma membrane is critical for the virulence of the human fungal pathogen Candida albicans . In addition to functioning as a protective barrier, the plasma membrane plays dynamic roles in a wide range of functions needed for virulence including nutrient uptake, cell wall synthesis, morphogenesis, resistance to stress, and invasive hyphal growth. Screening a collection of C. albicans mutants identified an understudied gene that is important for invasive hyphal growth, which we have termed CWR1 (Cell Wall Regulatory kinase). A mutant strain lacking CWR1 displayed defects in resisting stressful conditions that exacerbate cell wall defects. The Cwr1 protein shows strong similarity to protein kinases, suggesting it plays a regulatory role in coordinating plasma membrane and cell wall functions. A Cwr1-green fluorescent protein (GFP) fusion protein localized to punctate patches associated with the plasma membrane that partially overlapped Membrane Compartment of Can1 (MCC)/eisosome domains. In contrast to the static MCC/eisosome domains, the Cwr1-GFP patches were very dynamic. Truncation mutants lacking C-terminal sequences distal to the protein kinase domain failed to show detectable localization at the plasma membrane. Surprisingly, these mutant strains did not show the defects of a cwr1Δ mutant, suggesting that localization to punctate patches associated with the plasma membrane is not essential for Cwr1 function. Altogether, these data indicate that Cwr1 contributes to the regulation of plasma membrane functions that promote proper morphogenesis and resistance to cell wall stress, both of which are important for C. albicans virulence., Importance: The ability of Candida albicans to grow invasively in the host and resist stress is critical for it to be an effective human pathogen. Identifying the genes that promote these processes is important for developing new strategies to block infection. Therefore, genetic methods were used in this study to identify a novel gene that is needed for invasive growth and stress resistance (Cell Wall Regulatory kinase [ CWR1 ]). Interestingly, the Cwr1 protein localized to punctate patches in the plasma membrane, some of which co-localized with specialized subdomains of the plasma membrane known as eisosomes that are known to promote stress resistance and invasive growth in the host. Thus, these studies identified a novel regulator of traits that are critical for C. albicans pathogenesis., Competing Interests: The authors declare no conflict of interest.

Candida glabrata is an opportunistic human fungal pathogen that causes superficial mucosal and life-threatening bloodstream infections in immunocompromised individuals. Remodeling in cell wall components has been extensively exploited by fungal pathogens to adapt to host-derived stresses, as well as immune evasion. How this process contributes to C. glabrata pathogenicity is less understood. Here, we applied RNA sequencing and an in vivo invasive infection model to elucidate the prompt response of C. glabrata during infection. Fungal transcriptomes show a dramatic alteration in the expression of Srp1/Tip1-family cell wall structural proteins during systemic infection. Deletion of all six genes in this family ( TIR2-5 and AWP6-7 ) that are upregulated during infection leads to a significantly lower fungal burden in organs, as well as an attenuated virulence in the dextran sulfate sodium-induced colitis model. The tir2-5 awp6-7 sextuple mutant does not display any defect in response to host-derived stresses. Rather, deletion of all these six genes results in a lower cell surface exposure of an adhesin Epa1, which could contribute to its reduced adhesion to epithelial cells and cytotoxicity, as well as attenuated virulence. Our study reveals that cell wall remodeling triggered by the alteration in the expression of structural proteins is a key virulence attribute in C. glabrata that facilitates this fungus adhering to host cells and persisting in organs.IMPORTANCE Candida glabrata is one of the most frequent causes of candidiasis after Candida albicans . While C. albicans has been extensively studied, the mechanisms of infection and invasion of C. glabrata have not been fully elucidated. Using an infection model of systemic candidiasis and RNA sequencing, we show that there is a dramatic change in the expression of Srp1/Tip1-family genes during infection. Deletion of all six Srp1/Tip1-family genes that are upregulated during infection decreases the amount of cell wall-localized Epa1, probably reflecting the reduced adherence to epithelial cells and attenuated virulence in the sextuple mutant. These data suggest that alterations in the expression of Srp1/Tip1-family structural proteins trigger cell wall remodeling that increases the cell surface exposure of adhesins, such as Epa1, to promote virulence. Our study provides a pathogenic mechanism associated with C. glabrata in ensuring its sustenance and survival during infection., Competing Interests: The authors declare no conflict of interest.

ATP-binding cassette (ABC) transporter proteins, one of the largest families of membrane transport proteins, participate in almost all biological processes and widely exist in living organisms. Microsporidia are intracellular parasites; they can reduce crop yields and pose a threat to human health. The ABC proteins are also present in microsporidia and play a critical role in their proliferation and energy transport. In this study, a novel ABC transporter protein of Nosema bombycis named NoboABCG1.3 was identified. The NoboABCG1.3 protein is comprised of 640 amino acids, which contain six transmembrane domains and one nucleotide-binding domain. After N. bombycis infection of cells or tissues, quantitative reverse transcription polymerase chain reaction analysis revealed a progressive elevation in the transcript levels of NoboABCG1.3. Downregulation of NoboABCG1.3 expression significantly inhibited N. bombycis proliferation. Subsequently, a transgenic cell line stably expressing an interfering fragment of NoboABCG1.3 was established, which exhibited extreme inhibition on the proliferation of N. bombycis. These findings indicate that NoboABCG1.3 plays a role in the proliferation of N. bombycis and holds promise as a target for developing N. bombycis-resistant silkworms., Competing Interests: Declarations. Ethics approval and consent to participate: This study received approval from the Laboratory Animals Ethics Review Committee of Southwest University (Chongqing, China), ensuring adherence to the Guidelines for Ethical Review of Experimental Animal Welfare (GBT35892-2018) throughout all animal experiments. Consent to participate: All authors confirm their participation in the study. Consent for publication: All authors consent to publication of the manuscript. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Fungi can synthesize a diverse range of melanins with appropriate physicochemical and biological characteristics for numerous applications in health, environmental protection, energy, and industry. Gaining deeper insights into the chemical structures, biosynthetic pathways, and regulatory mechanisms of fungal melanin would establish a basis for metabolic engineering approaches, aimed at enhancing production efficiency and creating custom-designed melanin with desirable material properties. Due to growing interest in their beneficial effects and applications, research on the structure, biosynthesis, and regulation of fungal melanin has significantly advanced. This review highlighted recent progress in fungal melanin production and applications, concentrating on structure, biosynthesis, and regulatory networks, and suggested how an improved understanding of melanin biosynthesis could enable efficient production for future applications., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: The complete mitochondrial respiratory chain is a precondition for maintaining cellular energy supply, development, and metabolic balance. Due to the evolutionary differentiation of complexes and the semi-autonomy of mitochondria, respiratory chain subunits have become critical targets for crop improvement and fungal control. In fungi, mitochondrial complex I mediates growth and metabolism. However, the role of this complex in the pathogenesis of phytopathogenic fungi is largely unknown., Results: In this study, we identified the NADH: ubiquinone oxidoreductase 24-kDa subunit (VdNuo1) of complex in vascular wilt pathogen, Verticillium dahliae, and examined its functional conservation in phytopathogenic fungi. Based on the treatments with respiratory chain inhibitors, the mitochondria-localized VdNuo1 was confirmed to regulate mitochondrial morphogenesis and homeostasis. VdNuo1 was induced during the different developmental stages in V. dahliae, including hyphal growth, conidiation, and melanized microsclerotia development. The VdNuo1 mutants displayed variable sensitivity to stress factors and decreased pathogenicity in multiple hosts, indicating that VdNuo1 is necessary in stress tolerance and full virulence. Comparative transcriptome analysis demonstrated that VdNuo1 mediates global transcriptional effects, including oxidation and reduction processes, fatty acid, sugar, and energy metabolism. These defects are partly attributed to impairments of mitochondrial morphological integrity, complex assembly, and related functions. Its homologue (CgNuo1) functions in the vegetative growth, melanin biosynthesis, and pathogenicity of Colletotrichum gloeosporioides; however, CgNuo1 does not restore the VdNuo1 mutant to normal phenotypes., Conclusions: Our results revealed that VdNuo1 plays important roles in growth, metabolism, microsclerotia development, stress tolerance, and virulence of V. dahliae, sharing novel insight into the function of complex I and a potential fungicide target for pathogenic fungi., Competing Interests: Declarations. Ethics approval and consent to participate: This article does not contain any experiments with animals or human participants that were performed by the contributing authors. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Alternative splicing (AS) is a key element of eukaryotic gene expression that increases transcript and proteome diversity in cells, thereby altering their responses to external stimuli and stresses. While AS has been intensively researched in plants and animals, its frequency, conservation, and putative impact on virulence, are relatively still understudied in plant pathogenic fungi. Here, we profiled the AS events occurring in genes of Cladosporium fulvum isolates Race 5 and Race 4, during nearly a complete compatible infection cycle on their tomato host. Our studies revealed extensive heterogeneity in the transcript isoforms assembled from different isolates, infections, and infection timepoints, as over 80% of the transcript isoforms were singletons that were detected in only a single sample. Despite that, nearly 40% of the protein-coding genes in each isolate were predicted to be recurrently AS across the disparate infection timepoints, infections, and the two isolates. Of these, 37.5% were common to both isolates and 59% resulted in multiple protein isoforms, thereby putatively increasing proteome diversity in the pathogen by 31% during infections. An enrichment analysis showed that AS mostly affected genes likely to be involved in the transport of nutrients, regulation of gene expression, and monooxygenase activity, suggesting a role for AS in finetuning adaptation of C. fulvum on its tomato host during infections. Tracing the location of the AS genes on the fungal chromosomes showed that they were mostly located in repeat-rich regions of the core chromosomes, indicating a causal connection between gene location on the genome and propensity to AS. Finally, multiple cases of differential isoform usage in AS genes of C. fulvum were identified, suggesting that modulation of AS at different infection stages may be another way by which pathogens refine infections on their hosts., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zaccaron et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Candida albicans is a member of the human commensal microbiota but can also cause opportunistic infections, including life-threatening invasive candidiasis, particularly in immunocompromised patients. One of the important features of C. albicans commensalism and virulence is its ability to adapt to diverse environmental stress conditions within the host. Rap1 is a DNA-binding protein identified in yeasts, protozoa, and mammalian cells, and it plays multiple functions, including telomere regulation. Intriguingly, our previous study showed that Rap1 is also involved in cell wall integrity, biofilm formation, and virulence in C. albicans. In this work, using RNA-seq analysis and other approaches, the role of C. albicans Rap1 in oxidative stress response was further revealed. The RAP1-deletion mutant exhibited greater resistance to the superoxide generator menadione, a lower level of intracellular reactive oxygen species (ROS) upon menadione treatment, and higher expression levels of superoxide dismutase genes, all in response to oxidative stress. Moreover, the association between Rap1-mediated oxidative stress response and the mitogen-activated protein kinase (MAPK) Hog1, the transcription factor Cap1 and the TOR signalling was also determined. Together, these findings expand our understanding of the complex signalling and transcriptional mechanisms regulating stress responses in C. albicans., (© 2024 The Author(s).)

RNA-binding proteins (RBPs) play critical cellular roles by mediating various stages of RNA life cycles. Ssd1, an RBP with pleiotropic effects, has been implicated in aneuploidy tolerance in Saccharomyces cerevisiae but its mechanistic role remains unclear. Here, we used a network-based approach to inform on Ssd1's role in aneuploidy tolerance, by identifying and experimentally perturbing a network of RBPs that share mRNA targets with Ssd1. We identified RBPs whose bound mRNA targets significantly overlap with Ssd1 targets. For 14 identified RBPs, we then used a genetic approach to generate all combinations of genotypes for euploid and aneuploid yeast with an extra copy of chromosome XII, with and without SSD1 and/or the RBP of interest. Deletion of 10 RBPs either exacerbated or alleviated the sensitivity of wild-type and/or ssd1 Δ cells to chromosome XII duplication, in several cases indicating genetic interactions with SSD1 in the context of aneuploidy. We integrated these findings with results from a global overexpression screen that identified genes whose duplication complements ssd1 Δ aneuploid sensitivity. The resulting network points to a subgroup of proteins with shared roles in translational repression and P-body formation, implicating these functions in aneuploidy tolerance. Our results reveal a role for new RBPs in aneuploidy tolerance and support a model in which Ssd1 mitigates translation-related stresses in aneuploid cells., (© 2025 Dutcher and Gasch; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The FLO genes in Saccharomyces cerevisiae are repressed by heterochromatin formation, involving histone deacetylases, transcription factors and non-coding RNAs. Here, we report that mutations in the processivity factor POL30 (PCNA) that show transient derepression at the subtelomeres and the mating-type loci do not derepress FLO loci. However, deletions of the replisome stability factors RRM3 and TOF1 along with pol30 mutations induced flocculation phenotypes. The phenotypes correlated with increased expression of reporter proteins driven by the FLO11 promoter, the frequency of silent to active conversions of FLO11, and reduced expression of the regulatory long non-coding RNAs ICR1 and PWR1. Alterations in the local replication landscape of FLO11 indicate a link between defects in the fork protection complex and the stability of gene silencing. Analyses of these mutants at the subtelomeres and the HMLα locus showed a similar derepression phenotype and suggest transient instability of both active and silent states of FLO11. We conclude that RRM3 and TOF1 interact differentially with the pol30 mutations to promote transient derepression or complete epigenetic conversions of FLO11. We suggest that the interaction between POL30, RRM3 and TOF1 is essential to maintain epigenetic stability at the studied loci., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The filamentous fungus Penicillium verruculosum (anamorph Talaromyces verruculosus ) has been shown to be an efficient producer of secreted cellulases, used in biorefinery processes. Understanding the mechanisms of regulation of cellulase gene expression in the fungus P. verruculosum is a current task in industrial biotechnology, since it allows for targeted changes in the composition of the complex secreted by the fungus. Expression of cellulase genes in fungi is regulated mainly at the level of transcription via pathway-specific transcription factors (TF), the majority of which belong to the Zn(II)2Cys6 family of zinc binuclear cluster proteins. Transcriptional regulation of cellulase genes may have a species-specific pattern and involves several transcription factors. In this study, we used a qPCR method and transcriptome analysis to investigate the effect of knockouts and constitutive expression of genes encoding homologues of the regulatory factors XlnR and ClrB from P. verruculosum on the transcription of cbh1 , egl2 , and bgl1 genes, encoding three key cellulases, cellobiohydrolase, endoglucanase, and β-glucosidase, in the presence of various inducers. We have shown that the transcription factor XlnR of the filamentous fungus P. verruculosum is strictly responsible for the transcription of the main cellulolytic genes ( cbh1 , egl2 , and bgl1 ) in the presence of xylose and xylobiose, but not in the presence of cellobiose. ClrB/Clr-2, a homologue from P. verruculosum , does not represent the main transcription factor regulating transcription of cellulolytic genes in the presence of selected inducers, unlike in the cases of Aspergillus nidulans , Aspergillus niger , and Penicillium oxalicum; apparently, it has a different function in fungi from the genus Talaromyces . We have also shown that constitutive expression of the transcription factor XlnR resulted in 3.5- and 2-fold increases in the activity of xylanase and β-glucosidase in a B1-XlnR enzyme preparation, respectively. In a practical sense, the obtained result can be used for the production of enzyme preparations based on the P. verruculosum B1-XlnR strain used for the bioconversion of renewable cellulose-containing raw materials into technical sugars.

Nuclear pore proteins control nucleocytoplasmic transport; however, certain nucleoporins play regulatory roles in activities such as transcription and chromatin organization. The fission yeast basket nucleoporin Nup211 is implicated in mRNA export and is essential for cell viability. Nup211 preferentially associates with heterochromatin, however, it is unclear whether it plays a role in regulating transcription. To better understand its functions, we constructed a nup211 "shut-off" strain and observed that Nup211 depletion led to severe defects in cell cycle progression, including septation and cytokinesis. Using RNA-Seq and RT-qPCR, we revealed that loss of Nup211 significantly altered the mRNA levels of a set of genes crucial for cell division. Using domain analysis and CRISPR/cas9 technology, we determined that the first 655 residues of Nup211 are sufficient for viability. This truncated protein was detected at the nuclear periphery. Furthermore, exogenous expression of this domain in nup211 shut-off cells effectively restored both cell morphology and transcript abundance for some selected genes. Our findings unveil a novel role for Nup211 in regulating gene expression., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kamel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fusarium venenatum is being extensively utilized for microbial protein production. However, its high dietary fiber content results in substantial carbon loss. Inhibition of chitin biosynthesis presents a promising strategy to improve the mycoprotein yield. Through transcriptomic and bioinformatics analyses, chitin synthase gene FvChs3 was identified as crucial for chitin synthesis in F. venenatum . Knockout of the FvChs3 gene resulted in mycelial expansion and a 26% reduction in the chitin content of strain Δ FvChs3 . Ethanol production from fermentation decreased by 47%, while the carbon conversion efficiency and protein conversion increased by 16% and 36%, respectively. Transcriptomic analysis revealed an upregulation of nitrogen metabolism in Δ FvChs3 , while genes related to the glycolysis pathway for ethanol synthesis were downregulated. Further knockout of pyruvate decarboxylase gene FvPDC6 in Δ FvChs3 accelerated growth, leading to improvements in carbon and protein conversion of 29% and 40%, respectively. This research lays the foundation for enhancing fungal protein production.

Transcriptional rewiring generates phenotypic novelty, acting as an important mechanism contributing to evolutionary development, speciation, and adaptation in all organisms. The phenotypic outcomes (functions) of transcription factor (TF) activity are determined by the combined effects of all target genes in the TF's regulatory network. Plastic rewiring of target genes accumulates during species divergence and ultimately alters phenotypes, indicating a TF functional switch. We define this phenomenon as 'disruptive rewiring', where the rewiring process disrupts the link between a TF and its original target genes that determine phenotypes. Here, we investigate if 'complete' disruptive rewiring is a prerequisite for a TF functional switch by employing chromatin immunoprecipitation sequencing, RNA expression, and phenotypic assays across yeast species. In yeasts where Sef1 targets TCA (tricarboxylic acid) cycle genes, we demonstrate that Sef1 orthologs can promote and inhibit respiratory growth by modulating the moonlighting function of their conserved target, NDE1. This modulation occurs without changing the overall association of Sef1 with TCA cycle genes. We propose that phenotypic masking by NDE1 promotes 'deceptive' disruptive rewiring of the Sef1 regulatory network in Saccharomyces cerevisiae, thereby potentially constraining future evolutionary trajectories., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Prions represent epigenetic regulator proteins that can self-propagate their structure and confer their misfolded structure and function on normally folded proteins. Like the mammalian prion PrPSc, prions also occur in fungi. While a few prions, like Swi1, affect gene expression, none are shown to affect heterochromatin structure and function. In fission yeast and metazoans, histone methyltransferase Clr4/Suv39 causes H3-Lys9 methylation, which is bound by the chromodomain protein Swi6/HP1 to assemble heterochromatin. Earlier, we showed that sng2-1 mutation in the Cut4 subunit of anaphase-promoting complex abrogates heterochromatin structure due to defective binding and recruitment of Swi6. Here, we demonstrate that the Cut4p forms a non-canonical prion form, designated as [SNG2], which abrogates heterochromatin silencing. [SNG2] exhibits various prion-like properties, e.g. non-Mendelian inheritance, requirement of Hsp proteins for its propagation, de novo generation upon cut4 overexpression, reversible curing by guanidine, cytoplasmic inheritance and formation of infectious protein aggregates, which are dissolved upon overexpression of hsp genes. Interestingly, [SNG2] prion imparts an enhanced tolerance to stress conditions, supporting its role in promoting cell survival under environmental stress during evolution., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Heterochromatin plays a critical role in regulating gene expression and maintaining genome integrity. While structural and enzymatic components have been linked to heterochromatin establishment, a comprehensive view of the underlying pathways at diverse heterochromatin domains remains elusive. Here, we developed a systematic approach to identify factors involved in heterochromatin silencing at pericentromeres, subtelomeres and the silent mating type locus in Schizosaccharomyces pombe. Using quantitative measures, iterative genetic screening and domain-specific heterochromatin reporters, we identified 369 mutants with different degrees of reduced or enhanced silencing. As expected, mutations in the core heterochromatin machinery globally decreased silencing. However, most other mutants exhibited distinct qualitative and quantitative profiles that indicate heterochromatin domain-specific functions, as seen for example for metabolic pathways affecting primarily subtelomere silencing. Moreover, similar phenotypic profiles revealed shared functions for subunits within complexes. We further discovered that the uncharacterized protein Dhm2 plays a crucial role in heterochromatin maintenance, affecting the inheritance of H3K9 methylation and the clonal propagation of the repressed state. Additionally, Dhm2 loss resulted in delayed S-phase progression and replication stress. Collectively, our systematic approach unveiled a landscape of domain-specific heterochromatin regulators controlling distinct states and identified Dhm2 as a previously unknown factor linked to heterochromatin inheritance and replication fidelity., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)