New protein-coding genes can evolve from previously noncoding genomic regions through a process known as de novo gene emergence. Evidence suggests that this process has likely occurred throughout evolution and across the tree of life. Yet, confidently identifying de novo emerged genes remains challenging. Ancestral sequence reconstruction is a promising approach for inferring whether a gene has emerged de novo or not, as it allows us to inspect whether a given genomic locus ancestrally harbored protein-coding capacity. However, the use of ancestral sequence reconstruction in the context of de novo emergence is still in its infancy and its capabilities, limitations, and overall potential are largely unknown. Notably, it is difficult to formally evaluate the protein-coding capacity of ancestral sequences, particularly when new gene candidates are short. How well-suited is ancestral sequence reconstruction as a tool for the detection and study of de novo genes? Here, we address this question by designing an ancestral sequence reconstruction workflow incorporating different tools and sets of parameters and by introducing a formal criterion that allows to estimate, within a desired level of confidence, when protein-coding capacity originated at a particular locus. Applying this workflow on ∼2,600 short, annotated budding yeast genes (<1,000 nucleotides), we found that ancestral sequence reconstruction robustly predicts an ancient origin for the most widely conserved genes, which constitute \"easy\" cases. For less robust cases, we calculated a randomization-based empirical P-value estimating whether the observed conservation between the extant and ancestral reading frame could be attributed to chance. This formal criterion allowed us to pinpoint a branch of origin for most of the less robust cases, identifying 49 genes that can unequivocally be considered de novo originated since the split of the Saccharomyces genus, including 37 Saccharomyces cerevisiae-specific genes. We find that for the remaining equivocal cases we cannot rule out different evolutionary scenarios including rapid evolution, multiple gene losses, or a recent de novo origin. Overall, our findings suggest that ancestral sequence reconstruction is a valuable tool to study de novo gene emergence but should be applied with caution and awareness of its limitations.

Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.

Peanuts (Arachis hypogaea L.) are among the most important leguminous crops in Argentina. During the growing season, they are frequently attacked by fungal diseases, including Thecaphora frezii. The spores of T. frezii are structures that confer resistance to this phytopathogen. The transition from teliospore to hypha is a characteristic process of some fungi, which is essential for completing their life cycle. Using the transcriptomes of teliospores and hyphae of T. frezii, we aimed to identify genes that were differentially expressed during this transition, and we found 134 up-regulated and 66 down-regulated genes, which would participate in different cellular processes such as: (a) cell cycle and DNA processing; (b) cell fate; (c) rescue, defense and cellular virulence; (d) detoxification by CYP450; (e) energy; (f) nutrient interaction and nutritional adaptation; (g) metabolism; (g) proteins with binding functions or cofactor requirements; (h) stress, cell differentiation and biogenesis of cell components; and (i) transport, cell communication and transcription. The identification of genes in T. frezii and their expression levels during different stages of differentiation could contribute to our understanding of the biological mechanisms in this fungus.

Lignin has significant potential as an abundant and renewable source for commodity chemicals yet remains vastly underutilized. Efforts towards engineering a biochemical route to the valorization of lignin are currently limited by the lack of a suitable heterologous host for the production of lignin-degrading enzymes. Here, we show that expression of fungal genes in Nicotiana benthamiana enables production of members from seven major classes of enzymes associated with lignin degradation (23 of 35 tested) in soluble form for direct use in lignin activity assays. We combinatorially characterized a subset of these enzymes in the context of model lignin dimer oxidation, revealing that fine-tuned coupling of peroxide-generators to peroxidases results in more extensive C-C bond cleavage compared to direct addition of peroxide. Comparison of peroxidase isoform activity revealed that the extent of C-C bond cleavage depends on peroxidase identity, suggesting that peroxidases are individually specialized in the context of lignin oxidation. We anticipate the use of N. benthamiana as a platform to rapidly produce a diverse array of fungal lignin-degrading enzymes will facilitate a better understanding of their concerted role in nature and unlock their potential for lignin valorization, including within the plant host itself.

BACKGROUND: A wide range of frequency of azole-resistance in A fumigatus in different patient populations worldwide was observed threatening to reduce therapeutic options.
OBJECTIVE: Estimate the prevalence of azole-resistance, investigate the molecular mechanisms of resistance, compare the genotypes of resistant clinical isolates with those from the surrounding environment.
METHODS: Aspergillus isolates were collected by seven Italian hospital microbiology laboratories. Strains were isolated from different clinical samples from unselected patients. The azole-resistance was evaluated using screening test and microdilution EUCAST method. The molecular mechanism of resistance was performed sequencing the cyp51A gene. Resistant isolates were genotyped by microsatellite analysis and their profiles compared with those of azole-resistant isolates from previous Italian studies.
RESULTS: 425 Aspergillus isolates from 367 patients were analysed. The azole-resistance rates were 4.9% and 6.6% considering all Aspergillus spp. isolates and the A fumigatus sensu stricto, respectively. All resistant isolates except one were from a single hospital. Two rare azole-resistant species were identified: A thermomutatus and A lentulus. The predominant resistance mechanism was TR34 /L98H. No correlation between the clinical resistant strains and environmental isolates from patients\' home/work/ward was observed. The analysis of the molecular correlation between the resistant clinical strains collected in the present study and those of environmental and clinical origin collected in previous Italian studies reveals a progressive diversification of azole-resistant genotypes starting from a founder azole-resistant genotype.
CONCLUSIONS: This study confirms the trend of azole-resistance rate in Italy, showing a geographical difference. Data reinforce the importance of surveillance programmes to monitor the local epidemiological situation.

BACKGROUND: Despite the latest advancements in metabolic engineering for genome editing and characterization of host performance, the successful development of robust cell factories used for industrial bioprocesses and accurate prediction of the behavior of microbial systems, especially when shifting from laboratory-scale to industrial conditions, remains challenging. To increase the probability of success of a scale-up process, data obtained from thoroughly performed studies mirroring cellular responses to typical large-scale stimuli may be used to derive crucial information to better understand potential implications of large-scale cultivation on strain performance. This study assesses the feasibility to employ a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime and aims to understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance.
RESULTS: We find that mutant population diversity is maintained through multiple seed trains, enabling large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes tested in this study, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that all encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Ecological analysis of beta diversity between all samples revealed significant population divergence over time and showed feed specific consequences of population structure. Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant stresses. Our observations indicate that, for this yeast deletion collection, the selection of the feeding scheme which affects the accumulation of the fermentative by-product ethanol impacts the diversity of the mutant pool to a higher degree as compared to the pH of the culture broth. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses.
CONCLUSIONS: Our results demonstrate the feasibility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions and to understand critical stages of a scale-up process where variability emerges, and selection pressure gets imposed. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.

The Roux-en-Y gastric bypass (RYGB) remains the most effective treatment for morbidly obese patients to lower body weight and improve glycemic control. There is recent evidence that the mycobiome (fungal microbiome) can aggravate disease severity in a number of diseases including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and hepatitis; moreover, a dysbiotic fungal microbiota has been reported in the obese. We characterized fungal and bacterial microbial composition in fecal samples of 16 morbidly obese patients before and three months after RYGB surgery and compared with nine healthy controls. We found that RYGB surgery induced a clear alteration in structure and composition of the gut fungal and bacterial microbiota. Beta diversity analysis revealed significant differences in bacterial microbiota between obese patients before surgery and healthy controls (P < 0.005) and a significant, unidirectional shift in RYGB patients after surgery (P < 0.001 vs. before surgery). In contrast, there was no significant difference in fungal microbiota between groups but individually specific changes after RYGB surgery. Interestingly, RYGB surgery induced a significant reduction in fungal alpha diversity namely Chao1, Sobs, and Shannon diversity index (P<0.05, respectively) which contrasts the trend for uniform changes in bacteria towards increased richness and diversity post-surgery. We did not observe any inter-kingdom relations in RYGB patients but in the healthy control cohort and there were several correlations between fungi and bacteria and clinical parameters (P<0.05, respectively) that warrant further research. Our study identifies changes in intestinal fungal communities in RYGB patients that are distinct to changes in the bacterial microbiota.

Conidiobolus spp. are important saprophytic basal fungi. However, to date, no genomic-level data for decaying plant materials in the genus Conidiobolus has been reported. Here, we report that the 33.4-Mb genome of Conidiobolus heterosporus encodes 10,857 predicted genes. Conidiobolus heterosporus harbors 394 CAZyme-encoding genes belonging to 4 major modules but does not encode a polysaccharide lyase (PL). Many carbohydrate esterases (CEs) belonging to the family CE12 play crucial roles as pectin acetylesterases, and 14 genes were upregulated in the IM (fungus grown on inducing medium) among 17 expressed CE12 family genes. In addition, most of the genes in the GH132 CAZyme family showed a greater than 5-fold increase in expression in the IM compared with that in the wild type. Furthermore, 122 P450-encoding genes grouped into 11 families were detected in the fungal genome, most of which belonged to the CYP547 family (36 genes) followed by CYP548 (27 genes) and CYP5856 (25 genes). Interestingly, members of the families CYP5014 and CYP5136 were identified, the first time such enzymes have been described in a fungus. Our findings provide new insights into the genomics and genomic features of the saprophytic basal fungus C. heterosporus.Key Points• Genome of the saprobiotic basal fungus C. heterosporus was sequenced and analyzed.• 394 CAZymes but no PL family genes were found and expression levels were determined.• CE12 and GH132 proteins may play roles in the pectin and plant material degradation.• A large number of P450s but few P450 families existed in the fungus.

Network representations are flat while mechanisms are organized into a hierarchy of levels, suggesting that the two are fundamentally opposed. I challenge this opposition by focusing on two aspects of the ways in which large-scale networks constructed from high-throughput data are analysed in systems biology: identifying clusters of nodes that operate as modules or mechanisms and using bio-ontologies such as gene ontology (GO) to annotate nodes with information about where entities appear in cells and the biological functions in which they participate. Of particular importance, GO organizes biological knowledge about cell components and functions hierarchically. I illustrate how this supports mechanistic interpretation of networks with two examples of network studies, one using epistatic interactions among genes to identify mechanisms and their parts and the other using deep learning to predict phenotypes. As illustrated in these examples, when network research draws upon hierarchical information such as provided by GO, the results not only can be interpreted mechanistically but provide new mechanistic knowledge. This article is part of the theme issue \'Unifying the essential concepts of biological networks: biological insights and philosophical foundations\'.

Species of Endogonaceae (Endogonales, Mucoromycotina) are characterized by the formation of relatively large sporocarps and zygosporangia. Numerous species in this family remain undescribed or have unclear phylogenetic positions. In Asia specifically, the species diversity of this family is almost completely unknown. However, many mycobionts of bryophytes belonging to several novel clades in Endogonaceae have recently been identified phylogenetically. Therefore, establishing a robust taxonomic system for this family is essential. We obtained numerous sporocarps of undescribed Endogonaceae-like species from the Japanese islands. Morphological observation and multilocus phylogenetic analysis of nuc 18S rDNA (18S), nuc 28S rDNA (28S), and portions of two nuclear protein-coding regions-translation elongation factor 1-alpha (tef1) and RNA polymerase II large subunit (rpb1)-from these species resulted in the description of one new species each of Endogone and Jimgerdemannia and two new species of Vinositunica, gen. nov. Because Vinositunica is characterized by purplish sporocarps and red-wine-colored chlamydospores up to 700 μm in diameter, we emended the definition of Endogonaceae.