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Although fungi lack adenosine deaminase acting on RNA (ADAR) enzymes, adenosine to inosine (A-to-I) RNA editing was reported recently in Fusarium graminearum during sexual reproduction. In this study, we profiled the A-to-I editing landscape and characterized its functional and adaptive properties in the model filamentous fungus Neurospora crassa. A total of 40,677 A-to-I editing sites were identified, and approximately half of them displayed stage-specific editing or editing levels at different sexual stages. RNA-sequencing analysis with the Δstc-1 and Δsad-1 mutants confirmed A-to-I editing occurred before ascus development but became more prevalent during ascosporogenesis. Besides fungal-specific sequence and secondary structure preference, 63.5% of A-to-I editing sites were in the coding regions and 81.3% of them resulted in nonsynonymous recoding, resulting in a significant increase in the proteome complexity. Many genes involved in RNA silencing, DNA methylation, and histone modifications had extensive recoding, including sad-1, sms-3, qde-1, and dim-2. Fifty pseudogenes harbor premature stop codons that require A-to-I editing to encode full-length proteins. Unlike in humans, nonsynonymous editing events in N. crassa are generally beneficial and favored by positive selection. Almost half of the nonsynonymous editing sites in N. crassa are conserved and edited in Neurospora tetrasperma. Furthermore, hundreds of them are conserved in F. graminearum and had higher editing levels. Two unknown genes with editing sites conserved between Neurospora and Fusarium were experimentally shown to be important for ascosporogenesis. This study comprehensively analyzed A-to-I editing in N. crassa and showed that RNA editing is stage-specific and generally adaptive, and may be functionally related to repeat induced point mutation and meiotic silencing by unpaired DNA.

Lineage-specific genes (LSGs) have long been postulated to play roles in the establishment of genetic barriers to intercrossing and speciation. In the genome of Neurospora crassa , most of the 670 Neurospora LSGs that are aggregated adjacent to the telomeres are clustered with 61% of the HET-domain genes, some of which regulate self-recognition and define vegetative incompatibility groups. In contrast, the LSG-encoding proteins possess few to no domains that would help to identify potential functional roles. Possible functional roles of LSGs were further assessed by performing transcriptomic profiling in genetic mutants and in response to environmental alterations, as well as examining gene knockouts for phenotypes. Among the 342 LSGs that are dynamically expressed during both asexual and sexual phases, 64% were detectable on unusual carbon sources such as furfural, a wildfire-produced chemical that is a strong inducer of sexual development, and the structurally-related furan 5-hydroxymethyl furfural (HMF). Expression of a significant portion of the LSGs was sensitive to light and temperature, factors that also regulate the switch from asexual to sexual reproduction. Furthermore, expression of the LSGs was significantly affected in the knockouts of adv-1 and pp-1 that regulate hyphal communication, and expression of more than one quarter of the LSGs was affected by perturbation of the mating locus. These observations encouraged further investigation of the roles of clustered lineage-specific and HET-domain genes in ecology and reproduction regulation in Neurospora , especially the regulation of the switch from the asexual growth to sexual reproduction, in response to dramatic environmental conditions changes.

Neurospora crassa propagates through dissemination of conidia, which develop through specialized structures called conidiophores. Recent work has identified striking variation in conidiophore morphology, using a wild population collection from Louisiana, United States of America to classify 3 distinct phenotypes: Wild-Type, Wrap, and Bulky. Little is known about the impact of these phenotypes on sporulation or germination later in the N. crassa life cycle, or about the genetic variation that underlies them. In this study, we show that conidiophore morphology likely affects colonization capacity of wild N. crassa isolates through both sporulation distance and germination on different carbon sources. We generated and crossed homokaryotic strains belonging to each phenotypic group to more robustly fit a model for and estimate heritability of the complex trait, conidiophore architecture. Our fitted model suggests at least 3 genes and 2 epistatic interactions contribute to conidiophore phenotype, which has an estimated heritability of 0.47. To uncover genes contributing to these phenotypes, we performed RNA-sequencing on mycelia and conidiophores of strains representing each of the 3 phenotypes. Our results show that the Bulky strain had a distinct transcriptional profile from that of Wild-Type and Wrap, exhibiting differential expression patterns in clock-controlled genes (ccgs), the conidiation-specific gene con-6, and genes implicated in metabolism and communication. Combined, these results present novel ecological impacts of and differential gene expression underlying natural conidiophore morphological variation, a complex trait that has not yet been thoroughly explored.

The filamentous fungus Neurospora crassa, a model microbial eukaryote, has a life cycle with many features that make it suitable for studying experimental evolution. However, it has lacked a general tool for estimating relative fitness of different strains in competition experiments. To remedy this need, we constructed N. crassa strains that contain a modified csr-1 locus and developed an assay for detecting the proportion of the marked strain using a post PCR high resolution melting assay. DNA extraction from spore samples can be performed on 96-well plates, followed by a PCR step, which allows many samples to be processed with ease. Furthermore, we suggest a Bayesian approach for estimating relative fitness from competition experiments that takes into account the uncertainty in measured strain proportions. We show that there is a fitness effect of the mating type locus, as mating type mat a has a higher competitive fitness than mat A. The csr-1* marker also has a small fitness effect, but is still a suitable marker for competition experiments. As a proof of concept, we estimate the fitness effect of the qde-2 mutation, a gene in the RNA interference pathway, and show that its competitive fitness is lower than what would be expected from its mycelial growth rate alone.

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

Trimethylated lysine 27 on histone H3 (H3K27me3) is present in Drosophila, Arabidopsis. worms, and mammals, but is absent from yeasts that have been examined. We identified and analyzed H3K27me3 in the filamentous fungus Neurospora crassa and in other Neurospora species. H3K27me3 covers 6.8% of the N. crassa genome, encompassing 223 domains, including 774 genes, all of which are transcriptionally silent N. crassa H3K27me3-marked genes are less conserved than unmarked genes and only ~35% of genes marked by H3K27me3 in N. crassa are also H3K27me3-marked in Neurospora discreta and Neurospora tetrasperma. We found that three components of the Neurospora Polycomb repressive complex 2 (PRC2)—[Su-(var) 3-9; E(z); Trrthorax] (SET)-7, embryonic ectoderm development (EED), and SU(Z)12 (suppressor of zeste12)—are required for H3K27me3, whereas the fourth component Neurospora protein 55 (an N. crassa homolog of p55/RbAp48), is critical for H3K27me3 only at subtelomeric domains. Loss of H3K27me3, caused by deletion of the gene encoding the catalytic PRC2 subunit, set-7, resulted in up-regulation of 130 genes, including genes in both H3K27me3-marked and unmarked regions.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3 , found in the filamentous ascomycete Neurospora intermedia . Using long-read sequencing, we generated the first complete and well-annotated genome assemblies of large, highly diverged, non-recombining regions associated with meiotic drive elements. Phylogenetic analysis shows that, even though Sk-2 and Sk-3 are located in the same chromosomal region, they do not form sister clades, suggesting independent origins or at least a long evolutionary separation. We conclude that they have in a convergent manner accumulated similar patterns of tandem inversions and dense repeat clusters, presumably in response to similar needs to create linkage between genes causing drive and resistance. Meiotic drive elements are selfish genetic elements that mediate a skew in their sexual transmission from parent to offspring. Here, the authors sequence and analyze large and complex genomic regions associated with meiotic drive elements in the fungus Neurospora intermedia .

Fungal biotechnology can drive sustainability, offering eco-friendly solutions for energy, healthcare, nutraceuticals, and environmental challenges.The genomic and metabolic adaptability of fungi enables the efficient production of biofuels, enzymes, nutraceuticals, and other high-value bioproducts.Biosynthetic gene clusters in fungi support the discovery and production of essential secondary metabolites for pharmaceuticals and industry.Artificial intelligence-driven design–build–test–learn cycles accelerate fungal strain optimization, enhancing metabolic engineering and improving bioprocess efficiency. Fungal biotechnology plays a vital role in advancing sustainability by offering innovative solutions for resource efficiency, environmental protection, and health improvements. Fungal systems are highly adaptable compared with other biotechnologies, with unique genomic and metabolic functions that enable the large-scale production of valuable compounds. This review emphasizes how fungal biotechnology contributes to global sustainability goals, particularly through artificial intelligence (AI)-driven methods that accelerate strain optimization and metabolic engineering. Engineered Aspergillus strains, with enhanced enzyme production, and Neurospora, a model organism, demonstrate significant potential for industrial applications. These advancements offer cost-effective and resource-efficient solutions, underscoring the importance of interdisciplinary collaboration in fungal biology, genomics, enzymes, and computational approaches to scale fungal biotechnology for sustainable outcomes. Fungal biotechnology plays a vital role in advancing sustainability by offering innovative solutions for resource efficiency, environmental protection, and health improvements. Fungal systems are highly adaptable compared with other biotechnologies, with unique genomic and metabolic functions that enable the large-scale production of valuable compounds. This review emphasizes how fungal biotechnology contributes to global sustainability goals, particularly through artificial intelligence (AI)-driven methods that accelerate strain optimization and metabolic engineering. Engineered Aspergillus strains, with enhanced enzyme production, and Neurospora, a model organism, demonstrate significant potential for industrial applications. These advancements offer cost-effective and resource-efficient solutions, underscoring the importance of interdisciplinary collaboration in fungal biology, genomics, enzymes, and computational approaches to scale fungal biotechnology for sustainable outcomes.

A Neurospora meiotic drive element known as Spore killer-2 (Sk-2) achieves biased transmission through sexual reproduction by killing siblings that inherit a competing allele... Sk-2 is a meiotic drive element that was discovered in wild populations of Neurospora fungi over 40 years ago. While early studies quickly determined that Sk-2 transmits itself through sexual reproduction in a biased manner via spore killing, the genetic factors responsible for this phenomenon have remained mostly unknown. Here, we identify and characterize rfk-1, a gene required for Sk-2-based spore killing. The rfk-1 gene contains four exons, three introns, and two stop codons, the first of which undergoes RNA editing to a tryptophan codon during sexual development. Translation of an unedited rfk-1 transcript in vegetative tissue is expected to produce a 102-amino acid protein, whereas translation of an edited rfk-1 transcript in sexual tissue is expected to produce a protein with 130 amino acids. These findings indicate that unedited and edited rfk-1 transcripts exist and that these transcripts could have different roles with respect to the mechanism of meiotic drive by spore killing. Regardless of RNA editing, spore killing only succeeds if rfk-1 transcripts avoid silencing caused by a genome defense process called meiotic silencing by unpaired DNA (MSUD). We show that rfk-1’s MSUD avoidance mechanism is linked to the genomic landscape surrounding the rfk-1 gene, which is located near the Sk-2 border on the right arm of chromosome III. In addition to demonstrating that the location of rfk-1 is critical to spore-killing success, our results add to accumulating evidence that MSUD helps protect Neurospora genomes from complex meiotic drive elements.

Fungal fermentation of food and agricultural by-products holds promise for improving food sustainability and security. However, the molecular basis of fungal waste-to-food upcycling remains poorly understood. Here we use a multi-omics approach to characterize oncom, a fermented food traditionally produced from soymilk by-products in Java, Indonesia. Metagenomic sequencing of samples from small-scale producers in Western Java indicated that the fungus Neurospora intermedia dominates oncom. Further transcriptomic, metabolomic and phylogenomic analysis revealed that oncom-derived N. intermedia utilizes pectin and cellulose degradation during fermentation and belongs to a genetically distinct subpopulation associated with human-generated by-products. Finally, we found that N. intermedia grew on diverse by-products such as fruit and vegetable pomace and plant-based milk waste, did not encode mycotoxins, and could create foods that were positively perceived by consumers outside Indonesia. These results showcase the traditional significance and future potential of fungal fermentation for creating delicious and nutritious foods from readily available by-products. A multi-omics analysis of oncom, an Indonesian fermented food made from soymilk waste, shows how associated fungi break down food waste to yield nutritious and positively received foods.