Frontiers in Fungal Biology

Nakaseomyces glabratus is a globally distributed opportunistic fungal pathogen. An ongoing discussion in studies of N. glabratus population structure has been whether genetic clusters are best defined using multilocus sequence typing (MLST) or short-read whole-genome sequencing (WGS). To assess the concordance between MLST- and WGS-based phylogenies, we analyzed a dataset of 548 N. glabratus WGS sequences from 12 countries. Clusters identified from WGS largely recapitulated the MLST-defined sequence type (ST) groups: fourteen WGS clusters were composed of a single MLST ST, and the remaining contained STs with very closely related MLST profiles. We thus propose a pragmatic naming convention, consistent with the system used in other microbial species, which specifies WGS cluster labels based on the primary ST. From the large WGS isolate dataset, we determined the prevalence of admixture and genomic variants. Interestingly, seven of the nine singleton isolates were admixed, in addition to 58 isolates from six different clusters. Aneuploidy was detected in 4% of isolates, most commonly in chrE, which contains ERG11, the gene encoding the enzyme targeted by azole antifungals. Aneuploid chromosomes did not exhibit elevated heterozygosity relative to the sequencing error rate, consistent with instability of extra chromosome copies. Copy number variants were found in 3% of the isolates; some of the CNVs co-occurred with aneuploidies, and were primarily identified on chrD, chrE, chrI, and chrM. Our findings demonstrate that deep splits between clusters preserve the utility of MLST ST designations for clade-level designation, yet underscore the utility of WGS for high-resolution genomic analyses. Article SummaryThere is an ongoing debate in studies on Nakaseomyces glabratus about whether traditional MLST analysis is sufficient to determine population structure, or whether the precision of whole genome sequencing (WGS) is necessary. We analyzed WGS data from 548 isolates from around the world. We found a very strong agreement between the two methods. We propose a hybrid naming system, where cluster names are based on the dominant MLST group. We used the WGS data to show that admixed isolates, and those with extra chromosomes or CNVs are rare (<7% of isolates in each class) and are distributed throughout the phylogeny.

Peanut smut, caused by the fungus Thecaphora frezzii, is a significant disease of peanuts in Argentina. Infected plants have seeds replaced by a mass of dark teliospores, reducing yield and seed quality. To prevent the spread of the pathogen, several countries have limited import of raw peanuts from Argentina, a major grower and exporter. Following successful in vitro culture of the fungus in its haploid stage, we produced a chromosome-level genome assembly of the species for the first time. We compare this genome with those of 49 other species of true smut fungi, or Ustilaginomycetes, including species of medical, agricultural, and industrial importance, some of which are known as pathogens and others only as saprotrophic yeasts. At almost 39 Mb, T. frezzii has the largest genome of the smut fungi sequenced to date and the highest repetitive content. While it shares some core effectors with species of the distantly related and better studied Ustilago and related fungi, predicted effectors only found in T. frezzii or in Thecaphora suggest unique infection strategies. Comparisons among the 50 smut genomes also show that the 14 smut fungi observed only as yeasts share genomic traits such as low repeat content and generally smaller genomes. This supports the hypothesis that some smut fungi are adapted to saprotrophic growth as yeasts. The high-quality, annotated genome for T. frezzii will be a valuable resource for investigating the population dynamics and evolution of an economically important pathogen, as well as illuminating an understudied clade of smut fungi. Article summaryPeanut smut is a destructive and costly disease of peanuts in Argentina. For the first time, a high quality, annotated genome is presented for the causal agent, Thecaphora frezzii. This fungus has the largest and most repetitive genome of the true smut fungi, thus prompting comparison with 49 other species of smut fungi with available genomes, including non-pathogenic ones. While it shares some likely pathogenicity factors with well-studied smuts, it has many unique genes, a trait reflective of its evolutionary distance and likely novel mechanism for infection. This important genomic resource will benefit research regarding the evolution and adaption of T. frezzii, the development of diagnostic tools that enable rapid detection of it, and the study of smut fungi broadly.

Maudiozyma humilis is the second most frequently encountered yeast species in sourdough bread. Despite its ecological and food relevance, little is known about its evolutionary trajectories and phenotype traits of interest. Here we investigated the genomic and phenotypic diversity of a world-wide collection of 55 M. humilis strains, including 52 from sourdough, by combining genomic analysis, flow cytometry and high-throughput phenotyping of fermentation kinetics and fitness. Population genomic analysis revealed six genetically distinct clades, three diploid and three triploid, with no geographical or substrate-specific structuring. Phylogenetic, loss of heterozygosity (LOH) distribution and allele specific analysis indicated that triploid strains originated from both recent and more ancient hybridization events involving multiple diploid lineages. The absence of the HO gene, and mating-type silent cassettes, revealed that M. humilis is not able to carry mating-type switching. In addition, high linkage disequilibrium (LD) and variable LOH accumulation were observed consistent with a predominantly clonal reproduction. Last, an exceptionally high level of heterozygosity was detected, suggesting that occasional hybridization is the major driver of genetic diversity. Phenotypic characterization in a synthetic sourdough medium revealed variation in fermentation kinetics and fitness statistically associated with the genetic clades. Interestingly, genetic distance between clades and strains better explain the phenotypic variation than difference in ploidy. Altogether, our findings highlight the complex evolutionary history of M. humilis, shaped by hybridization and ploidy variation and reveal that historical contingency, more than ploidy, shapes the phenotypic landscape of this species. Beyond providing the first analysis of M. humilis evolution, our result challenges the hypothesis that increases in ploidy are necessarily beneficial in domesticated species.

Wheat take-all is a root disease which devastates crop yields, caused by the ascomycete fungus Gaeumannomyces tritici. The closely related root endophyte, G. hyphopodioides, has been found to induce local host defence responses which confer protection against take-all and reduce disease severity. Chancellor et al. (2024) investigated host transcriptional response to early colonisation by each of these two fungi. Using this RNA-seq dataset in conjunction with newly available Gaeumannomyces reference genomes, we have completed the picture by characterising the fungal transcriptional activity underpinning these different lifestyles. Even at early time points, their transcriptional profiles differ: G. hyphopodioides shows signs of transcriptional reprogramming between 4 and 5 days post inoculation (dpi), mirroring the wheat response, whereas G. tritici expression varied very little between these two time points despite progressing into the vasculature, instead exhibiting a stealthy expression profile dominated by gene downregulation at earlier time points. Moreover, GO term enrichment in this study identified a stress-response unique to G. hyphopodioides, which may explain the formation of its subepidermal vesicles (SEVs), putative resting structures that are a key difference between the pathogen and non-pathogen, alongside upregulation of many putative effectors and CAZymes. The enrichment of a key lignin-degrading CAZyme may contribute to the lack of stress-response identified in G. tritici, allowing fungal hyphae to overcome localised host lignification. These findings highlight the transcriptional basis of colonisation differences and are a step towards understanding how closely related fungi with different lifestyles modulate their interactions within a common host and tissue.

Ophiostomatoids are an ecological group of microfungi that commonly associate with bark and ambrosia beetles. As well as being insect symbionts, they play significant ecological roles as plant pathogens, and include species responsible for major forest tree diseases. Despite their ecological similarities, ophiostomatoids are distributed across two quite distantly related orders, the Microascales and Ophiostomatales. Historically, these fungi were considered a single natural group; however, molecular studies have revealed their independent origins and convergent ecological strategies. Previous phylogenetic studies of these fungi have typically focused on resolving taxonomic issues or understanding individual lifestyles, such as beetle-cultivated ambrosia lineages or vascular wilt pathogens. As a result, we lack a comprehensive phylogenetic framework that integrates dense species-level sampling with ecological data across both orders. Such frameworks are essential for understanding the broader phylogenetic and ecological context in which key fungal lifestyles have evolved. Here, we assembled and analysed all available sequence data for the Microascales and Ophiostomatales from seven widely used fungal marker loci to reconstruct a densely sampled phylogeny for each order. We evaluated locus performance and showed that whilst individual loci fail to resolve many taxa, concatenated datasets produce robust, well-supported topologies consistent with published genomic studies. By mapping ecological traits onto these trees, we show that lifestyle diversity and beetle associations are much more variable in the Microascales than in the Ophiostomatales, despite comparable species richness. Presenting both orders together provides a unique comparative perspective on the ecology and evolution of ophiostomatoids. As metabarcoding datasets of ophiostomatoids become increasingly common, this integrative framework can offer a valuable resource for environmental sequence identification and investigating fungal lifestyle switches, which in turn can support future biodiversity and ecology studies.

Cadophora luteo-olivacea is an ecologically versatile fungus associated with grapevine trunk diseases, yet the extent to which strains from different hosts and environments differ in genome composition, functional potential, and pathogenicity remains poorly understood. Here, we performed a comparative genomic analysis of 12 C. luteo-olivacea isolates recovered from grapevine, almond, apple, Crocus bulbs, soil, air, wastewater, and deep-sea sediment. Genome assemblies were highly complete (BUSCO >99%) and ranged from 46.94 to 50.70 Mbp. Pairwise average nucleotide identity (ANI) revealed a cohesive 11-strain group and one markedly divergent strain, CBS 266.93. Phylogenomic analysis based on 2,645 single-copy orthologs further showed that CBS 266.93 lies outside the main C. luteo-olivacea clade and forms a sister relationship with Cadophora malorum, indicating that its taxonomic placement warrants reassessment. Across the remaining strains, broad functional conservation was observed, including similar KOG profiles, extensive carbohydrate-active enzyme repertoires (798-849 genes per genome), and abundant biosynthetic gene clusters (26-35 per genome). Transposable element content varied substantially among strains (0.67-4.45% of genome), but this variation did not parallel overall functional profiles. All isolates colonized grapevine leaves in vitro, although lesion severity differed significantly among strains, indicating conserved plant-colonizing capacity with quantitative variation in aggressiveness. Small RNA profiling of inoculated grapevine leaves further revealed isolate-associated differences in host miRNA family expression, particularly for miR398, miR827, and miR156. Together, these results show that most C. luteo-olivacea strains share a conserved genomic framework compatible with plant colonization, while retaining lineage-and strain-level phenotypic and host-associated variation.

Modes of reproduction and sexual strategies strongly influence the genetic diversity and evolutionary potential of a species. The ascomycete fungus Clonostachys rosea is reported to be homothallic (sexually self-fertile), although a rapid decay of genome-wide linkage disequilibrium is also reported, something that is not in line with an obligate homothallic mode of reproduction. To investigate this phenomenon, we identified the mating-type (MAT1) locus in 66 genome-sequenced C. rosea strains under the hypothesis that each strain contains genes from both MAT1 idiomorphs. Eleven strains indeed contained both MAT1-1 and MAT1-2 genes, suggesting homothallism. However, most strains harboured either MAT1-1 or MAT1-2 genes and co-existed in North America, Europe and China, suggesting heterothallism. The MAT1 locus of heterothallic strains was highly conserved, and the linkage disequilibrium half decay distance was 1050 bp, suggesting sexual outcrossing. The presence of conserved MAT1-1 or MAT1-2 idiomorphs in strains of other Clonostachys species shows that heterothallism is likely the ancestral state. A phylogenetic analysis of 2800 single-copy orthologous genes revealed that homothallic and heterothallic strains separated in two well-supported clades, indicating a single lineage of homothallic C. rosea, likely originating in South America, followed by intercontinental dispersal. Homothallic C. rosea strains displayed higher nucleotide diversity than heterothallic strains, indicating a lack of outcrossing. This unique case of both homothallic and heterothallic lineages within the same species provides an opportunity to study the genomic consequences of selfing in very closely related strains.

Botrytis cinerea is a necrotrophic fungal pathogen that infects thousands of plant species. During infection, these diverse plant hosts produce different specialized metabolites that can inhibit pathogen growth and shape pathogen fitness. However, the genetic architecture of pathogen resistance toward individual host defense metabolites remains poorly understood. To address this question, we exposed 83 B. cinerea isolates to the metabolite linalool and quantified metabolic and structural responses. Exposure revealed extensive phenotypic diversity across isolates. Genome-wide association identified 101 genes of interest associated with membrane transport and stress response regulation. Genetic associations were stronger for morphological traits than for metabolic traits, suggesting that hyphal architecture may have a complex genetic architecture contributing to linalool resistance. Together, these results establish natural variation in linalool response and provide candidate loci for understanding how generalist pathogens respond to host-derived chemical defenses. Article SummaryTo understand how a generalist pathogen responds to host defenses, we asked how Botrytis cinerea responds to linalool, a widespread monoterpene involved in plant defense. We exposed 83 B. cinerea isolates to 1000 {micro}M of linalool for 72 hours and quantified metabolic traits (growth curves and growth dynamics over time) and morphological traits (hyphal network features). Using GWA, we linked phenotypic variation to genetic variants. Results indicate substantial natural variation in linalool resistance and distinct genetic architectures across trait classes: metabolic responses are driven by a relatively small number of loci with larger effects, whereas structural/morphological responses appear more polygenic.

Fungi, particularly ascomycetes, exhibit diverse ecological lifestyles, including endophytism, pathogenicity, and saprotrophy. Species of the genus Alternaria are taxonomically and ecologically diverse, yet the genomic determinants underlying different lifestyles remain poorly understood. Here, we investigate lifestyle-associated genomic variation in Alternaria atra using two newly collected isolates obtained as plant endophytes. We confirm their taxonomic identity and generate draft genome assemblies for both isolates. We assess their phenotypic behaviour under laboratory conditions and examine their genomic features alongside those of a previously published A. atra isolate described as pathogenic. Despite differing isolation histories, the endophytic and pathogenic isolates exhibit similar behaviour under laboratory conditions and possess highly comparable genomic repertoires, including predicted effector proteins, carbohydrate-active enzymes, and biosynthetic gene clusters. We detect no clear genomic signatures distinguishing endophytic and pathogenic origins or lifestyles. These findings suggest that A. atra harbours a shared genomic repertoire compatible with multiple ecological strategies, supporting a model of lifestyle plasticity rather than fixed genomic specialization. Our results add to growing evidence that genome content alone does not reliably predict ecological roles in ascomycete fungi.

Epimutations are changes in chromatin modifications, such as DNA methylation or histone modifications. Some of these epigenetic changes can be inherited for several generations, and they potentially contribute to evolutionary processes. Estimates of epimutation rates now exists in a few species, but the presence and function of epigenetic marks are not conserved across different species. To understand the properties of epimutations in fungi, we performed a mutation accumulation experiment with the filamentous fungus Neurospora crassa and investigated spontaneous changes in DNA methylation and trimethylation of lysine 9 on histone H3 (H3K9me3) in the mutation accumulation lines. We observed that centromeric regions are hotspots of spontaneous DNA methylation changes in N. crassa. In these hotspot regions, DNA methylation changes were transmitted across mitoses, but changes occurring in euchromatin were not maintained. The rate of DNA methylation changes was around 30 000 fold faster than the genetic mutation rate. We did not observe spontaneous changes in H3K9me3 that were transmitted across mitoses. Our results show that while spontaneous epimutations occur in this species, they occur predominantly in gene poor heterochromatic regions, so their impact for evolutionary adaptation may be limited.

Diversisporales comprises species with worldwide distribution that produce glomoid, otosporoid, or tricisporoid spores. The recent reorganization of the order by Oehl et al. (2016) recognizes two families, Diversisporaceae and Corymbiglomeraceae, comprising one and five genera, respectively. Several Glomeromycotan specimens collected in northern and southeastern Mexico and in French Polynesian atolls were characterized using both morphological and molecular analyses. Phylogenetic inference revealed that they represent new members in the Diversisporales, supporting the reorganization of the genus Redeckera into three independent lineages: Albocarpum gen. nov., with A. arenaceum sp. nov., A. leptohyphum sp. nov., and A. fulvum comb. nov., Pulvinocarpum pulvinatum gen. et comb. nov., and Redeckera, which retains five species, including R. varelae sp. nov. In addition, we described Melanocarpum mexicanum gen. et sp. nov. and Diversispora papillosa sp. nov. A broader phylogeny, based on eDNA sequences and representative of Diversisporales species, including the newly described taxa, further supported the split of Redeckera and suggested three additional clades likely corresponding to a new family and two new genera, awaiting the discovery of representative morphospecies to be formally described. Using eDNA sequences metadata, the occurrences of the newly described taxa were mapped, allowing to recognize distribution patterns, mostly in the pantropical zone, distinguish widespread and rare species, and suggest possible endemisms. Finally, the coexistence of species forming large sporocarps (A. fulvum and A. leptohyphum) alongside species forming spores in loose aggregates (A. arenaceum), prompted us to propose a possible sporulation dimorphism in Albocarpum, an argument previously raised to explain the nested placement of Corymbiglomus and Paracorymbiglomus within the Redeckera clade.

BACKGROUNDDuring host colonization, fungal plant pathogens secrete effector-like proteins that alter host cell physiology and target plant-associated microbes. However, rapid evolution and low sequence conservation hinder the study and characterization of these proteins. The fungus Zymoseptoria passerinii infects Hordeum spp. and includes lineages adapted to wild and domesticated barley. To date, the evolution of effector-like proteins in this species has not been addressed. RESULTSWe combined a set of protein structure-based and network analyses to unravel the secretome of Z. passerinii. We first compared AlphaFold2 and ESMFold predictions to establish the baseline for structural analyses. We identified 72 structural clusters in the secretome, revealing fold-level relationships across divergent sequences. We showed that effector-like proteins with host putative immune-interfering functions evolved from a limited group of protein folds, whereas proteins with predicted antimicrobial properties were distributed across fold groups. Physicochemical comparisons indicate that antimicrobial effectors predominantly emerged through amino acid replacements on common effector-enriched scaffolds in Z. passerinii, reconfiguring surface charge and electrostatics. We analyzed intra- and interspecific structural variation in selected effector-enriched families by comparing Z. passerinii proteins and homologs in the sister species Z. tritici. We describe high structural similarity in core folds, with local variation in loop and surface-exposed regions, consistent with fold stability still enabling functional diversification. CONCLUSIONSThe secretome of Z. passerinii is organized around common structural folds that support diverse biological roles, including host manipulation and host-associated microbial interactions. Conserved scaffolds combined with local physicochemical variation likely contribute to rapid adaptive evolution in Z. passerinii.

IntroductionThe use of Saccharomyces cerevisiae to ferment alcoholic beverages is an ancient tradition, with genetic evidence indicating origins in Neolithic Asia, although the domestication process of the species is not fully understood. Kveik is a group of traditional yeasts used in farmhouse brewing in western Norway preserved through generations of rural brewing practice. While recent studies have highlighted the distinctiveness of kveik, its precise phylogenetic position, genetic diversity, and domestication history remain unclear. ResultsWe performed whole-genome sequencing on 62 samples representing 25 unique Norwegian strains selected using cultural heritage criteria, and generated telomere-to-telomere (T2T) assemblies for representative isolates. Phylogenomic and population genetic analyses reveal that kveik forms a paraphyletic and early diverging group with respect to other domesticated S. cerevisiae strains. Most strains exhibit low within-strain diversity, strong geographic clustering, and little evidence of gene-flow or admixture. Mitochondrial genomes and Ty1 retrotransposon profiles corroborate this distinct lineage history. We further show that previously reported signals of gene flow between kveik and Asian fermentation strains are likely artifacts caused by population structure and selection. Divergence time estimates suggest that the common ancestor of beer, kveik, and other liquid-phase fermenting strains originated from ancestral populations 4,000 to 8,000 years ago. ConclusionKveik yeasts represent a relic of early S. cerevisiae domestication, shaped by ancient human practices, migrations, and the spread of agriculture. Our genomic resource sheds light on yeast evolution and domestication. They likely comprise some of the oldest domesticated lineages in continuous use until today, connecting endangered intangible cultural heritage to an early genetic origin.

Eukaryotic genomic DNA is packaged in the nucleus as chromatin - a DNA-protein aggregate regulating genome function, including transcription. Chromatin is classified as either active euchromatin or silent heterochromatin, with each marked by distinct histone post-translational modifications (PTMs). Chromatin composition also mediates genome organization, including how heterochromatin aggregates at the nuclear periphery while euchromatin localizes to the nucleus center. In fungi, heterochromatic loci cluster, including independent centromere and telomere clusters that form the Rabl chromosome conformation. However, it is unknown if chromatin composition and genome organization are conserved in closely related fungi, and how they are impacted by large-scale chromosomal rearrangements. Here, we examined differences in histone PTM deposition, gene expression, and genome organization in two yeast species from the order Pichiales, which diverged from the common ancestor shared with Saccharomyces cerevisiae more than 200 million years ago. We focused on Ogataea polymorpha, which is used for industrial protein production, and Ogataea haglerorum, an isolate of which harbors a translocation between chromosomes 1 and 6. We show that the enrichment of three activating PTMs - the trimethylation of lysine 4 of histone H3 (H3K4me3) and the acetylation of lysine 9 of histone H3 (H3K9ac) or lysine 16 of histone H4 (H4K16ac) - are similar genome-wide yet individual gene orthologs have distinct chromatin and expression patterns. While both Ogataea genomes organize into a Rabl conformation, the O. haglerorum translocation alters subtelomeric chromatin composition and expression of genes affected by the translocation. Our work highlights the genome function differences that occur on a microevolutionary scale.

Histoplasma spp. is a dimorphic fungal primary pathogen that infects people worldwide and frequently affects immunosuppressed patients. Previous studies have identified the AMY1 gene product, the -amylase Amy1p, as essential for -glucan production and virulence in Histoplasma capsulatum. We identified two new genes (AMY2 and AMY3) in the Histoplasma genome that encode putative -amylases and made mutants using CRISPR/Cas9 technology, followed by evaluation of their role in -glucan biosynthesis and virulence. We also searched for AMY gene copies in 19 fungal genomes with the goals of identifying orthologs for AMY2 and AMY3, and establishing how many AMY copies existed across different fungi. We found that the number and type of -amylases vary depending on the fungal species; that all -amylases related to Histoplasma Amy1p belong to the GH13_5 subfamily, and all orthologs related to Histoplasmas Amy2p and Amy3p belong to the GH13_1 subfamily. We performed phylogenetic analyses of the three paralogs and revealed that the Histoplasma AMY duplications are ancient. We further established Amy2 is an ortholog of Aspergillus niger AgtA, and Aspergillus nidulans AmyD, and that it is partially involved in Histoplasma -glucan biosynthesis and virulence, while Amy3p is an ortholog of Aspergillus flavus Amy1, and it is dispensable for -glucan biosynthesis and virulence.

Yeasts ability to invade surfaces has important implications for infections and food contamination. Invasive growth in yeast is influenced by genetic and environmental factors. In this exploratory study, we investigated the effects of sodium sulfide, gene deletions, and environmental conditions on the invasive behaviour of the wine yeast strain AWRI 796. Sodium sulfide enhanced invasion in the (parent) AWRI 796 strain under nitrogen-limiting conditions, although its effect was obscured by experimental variability and pre-culture conditions. Genetic factors had a major effect on the overall invasive phenotype, with deletion of key genes suppressing invasion. Most gene-deletion mutants did not significantly affect how the colony responded to sulfide. In addition to sulfide and genotype, environmental conditions also influenced invasive behaviour. The pre-2xSLAD pre-culture condition was best for detecting sulfide-induced growth, and later plate washing time and decreased nutrient levels enhanced invasiveness. Our experimental design and findings provide a framework for understanding the determinants of yeast invasiveness, which may inform future studies on filamentous yeast behaviour.

We present a comparative analysis of 13 yeasts available for mead (honey wine) fermentation, a source of Saccharomyces cerevisiae diversity that has not yet been analyzed in detail. Using genomic, phenotyping, and analytic methods, we show that currently available mead yeasts belong to various clades of the species, most commonly to the Commercial Wine clade (5 of 13 samples). Mead yeasts in this group displayed genome structure variations and occasional loss of killer activity, despite being closely related. Historic European and traditional African mead isolates with sequenced genomes were found not to be closely related to any contemporary mead yeast product. The 13 yeasts tested here displayed high variability in oenological characteristics and in aroma production. Maximum ethanol tolerance ranged from 15 to 22% v/v, however, the most tolerant strain produced lower ethanol levels and retained high fructose content in experimental meads. The most abundant aroma components produced in meads were ethyl acetate, ethyl caprylate, isoamyl alcohol, and ethyl caprate, with similar aroma profiles in members of the Commercial Wine clade, and pronounced differences among other yeasts. Our results contribute to the knowledge of Saccharomyces yeasts in various fermentation environments, adding mead to the list of alcoholic beverages with a known diversity of starter cultures. Our results may aid strain selection for honey wine fermentations and inspire strain improvement.

Extreme climatic events such as heat waves pose major challenges to species survival and have profound impacts on evolutionary processes. Plasticity is thought to buffer organismal stress, yet the molecular mechanisms underlying plastic responses remain poorly understood. In particular, the role of transcriptional plasticity and stress memory in responding to repeated stress events remains unresolved. Here, we experimentally exposed clonal populations of eight divergent Saccharomyces yeast species with different thermal tolerances to repeated heat waves. We compared their phenotypic and transcriptomic profiles after a few generations of mitotic growth, reflecting transcriptional changes. Warm-adapted species maintained higher growth than cold-adapted species across heat wave exposures. Thermo-generalist species showed intermediate outcomes with one species improving growth across repeated heat waves. To interpret transcriptomic results, we used a conceptual framework separating no-memory (gene expression independent from prior exposure) from memory-associated responses (expression modulated by prior exposure). No-memory responses showed conserved transcriptomic signatures of proteostasis induction, and reduced expression of ribosome biogenesis and translation upon repeated heat waves. Memory-associated responses were more rare and highly species-specific, showing opposite patterns of (de)sensitization in ribosomal and translational pathways in species at the two extremes of thermal tolerance. Together, our results show that thermal resilience can arise through alternative transcriptional changes and suggests that warm- and cold-temperature specialists adopt divergent gene regulatory strategies upon repeated heat waves. With climate change projections indicating more frequent and intense heat waves, understanding plastic responses across species with ecologically and genetically different backgrounds is crucial.

Changes in regulatory sequences controlling the timing and activity of gene products underlie much of natural phenotypic variation. Yet, what these changes are and how they impact gene expression remain largely unknown. To address this question, we investigated how transcriptional activity and homeostatic responsiveness of orthologous promoters of the metabolic gene TDH3 evolved among Saccharomyces yeast. We found that promoter expression level increased specifically in the S. cerevisiae lineage and that a substantial part of this increase was caused by genetic variants located between the well-characterized, conserved binding sites for two direct transcriptional regulators. These nucleotide changes altered the promoters expression levels while leaving the expression dynamics conserved. Further, the effects of these nucleotide changes were only seen in the presence of a third transcription factor, TYE7p, which is recruited by the other transcription factors through protein-protein interactions. These results suggest that the cis-regulatory changes act through their influence on the collective assembly/activation of the transcription factors, and that changes acting through such a mechanism can allow distinct parts of gene expression, such as expression level and dynamics, to be tuned separately.

To shed light into the biology of bacteria belonging to the genus Xylophilus, including the grapevine pathogen Xylophilus ampelinus, we scrutinized all available genomes of the genus for the presence of type III secretion and flagellar systems. We found three different flagellar systems in the genus, one of which was present in all twelve strains with good-quality genome sequences were available. The other two flagellar systems were only detected in one or two strains. We also identified two types of type III secretion systems, likely under control of the AraC-type transcriptional activator HrpX. One system with resemblance to systems from plant-pathogenic bacteria was only found in the grapevine pathogen. The other system was found in three strains of Xylophilus, all isolated from plant material. We predicted genes that are co-regulated with the type III secretion systems, as supported by the presence of strongly conserved HrpX-binding promoter elements. We identified about 40 type III effectors in the grapevine pathogen with homologs in plant pathogenic bacteria. In contrast, a rhododendron flower isolate had only two type III effector gene candidates with conserved HrpX-binding promoter elements but many genes without homologs beyond the species. Finally, we predicted and confirmed three novel effector candidates from X. ampelinus to contain a functional type III secretion signal using an AvrBs1 reporter approach. The presence of type III effectors suggests that effector-triggered immunity may exist in grapevine or non-host plants and that strategies targeting type III effectors for resistance engineering may contribute to suitable control measures.