ruffle fungi differentiate into subterranean fruiting bodies bearing spores sequestered in an inconspicuous globouslike mass of hyphae. Truffle-forming species have evolved in nearly every major group of fleshy fungi over 100 times independently within the Ascomycota and Basidiomycota and the majority of evolutionary transitions to a truffle morphology have occurred in lineages establishing ectomycorrhizal mutualistic symbiosis with plants 1. This pattern suggests that symbiosis has been a major driver in the evolution of truffle diversity. The evolution of the hypogeous lifestyle across a diversity of truffle lineages also suggests that the transition from epigeous to hypogeous fruiting is driven by strong selection for traits, for example pungent odours, that promote

Fungi are a large group of eukaryotes found in nearly all ecosystems. More than 250 fungal genomes have already been sequenced, greatly improving our understanding of fungal evolution, physiology, and development. However, for the Pezizomycetes, an early-diverging lineage of filamentous ascomycetes, there is so far only one genome available, namely that of the black truffle, Tuber melanosporum, a mycorrhizal species with unusual subterranean fruiting bodies. To help close the sequence gap among basal filamentous ascomycetes, and to allow conclusions about the evolution of fungal development, we sequenced the genome and assayed transcriptomes during development of Pyronema confluens, a saprobic Pezizomycete with a typical apothecium as fruiting body. With a size of 50 Mb and ∼13,400 protein-coding genes, the genome is more characteristic of higher filamentous ascomycetes than the large, repeat-rich truffle genome; however, some typical features are different in the P. confluens lineage, e.g. the genomic environment of the mating type genes that is conserved in higher filamentous ascomycetes, but only partly conserved in P. confluens. On the other hand, P. confluens has a full complement of fungal photoreceptors, and expression studies indicate that light perception might be similar to distantly related ascomycetes and, thus, represent a basic feature of filamentous ascomycetes. Analysis of spliced RNA-seq sequence reads allowed the detection of natural antisense transcripts for 281 genes. The P. confluens genome contains an unusually high number of predicted orphan genes, many of which are upregulated during sexual development, consistent with the idea of rapid evolution of sex-associated genes. Comparative transcriptomics identified the transcription factor gene pro44 that is upregulated during development in P. confluens and the Sordariomycete Sordaria macrospora. The P. confluens pro44 gene (PCON_06721) was used to complement the S. macrospora pro44 deletion mutant, showing functional conservation of this developmental regulator.

Botrytis cinerea, the gray mold fungus, is an important plant pathogen. Field populations are characterized by variability with regard to morphology, the mode of reproduction (conidiation or sclerotia formation), the spectrum of secondary metabolites (SM), and virulence. Natural variation in bcvel1 encoding the ortholog of Aspergillus nidulans VeA, a member of the VELVET complex, was previously shown to affect light-dependent differentiation, the formation of oxalic acid (OA), and virulence. To gain broader insight into the B. cinerea VELVET complex, an ortholog of A. nidulans LaeA, BcLAE1, a putative interaction partner of BcVEL1, was studied. BcVEL1 but not its truncated versions interacts with BcLAE1 and BcVEL2 (VelB ortholog). In accordance with the expected common as well as specific functions of BcVEL1 and BcLAE1, the deletions of both genes result in similar though not identical phenotypes. Both mutants lost the ability to produce OA, to colonize the host tissue, and to form sclerotia. However, mutants differ with regard to aerial hyphae and conidia formation. Genome-wide expression analyses revealed that BcVEL1 and BcLAE1 have common and distinct target genes. Some of the genes that are underexpressed in both mutants, e.g., those encoding SM-related enzymes, proteases, and carbohydrate-active enzymes, may account for their reduced virulence.

Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the full-length protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Δbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Δbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Δbcvel1 mutants to nutrient starvation conditions.

Many organisms use circadian clocks to adapt to daily changes in the environment. Major insights into the molecular mechanisms of circadian oscillators have been gained through studies of the model organism Neurospora crassa; however, little is known about molecular components of circadian clocks in other fungi. An important part of the N. crassa circadian clock is the frequency (frq) gene, homologs of which can be found in Sordariomycetes, Dothideomycetes, and Leotiomycetes, but not Eurotiomycetes. Recently, we identified a frq homolog in Pyronema confluens, a member of the early-diverging Pezizomycete lineage of filamentous ascomycetes. The P. confluens FRQ shares many conserved domains with the N. crassa FRQ. However, there is no known morphological phenotype showing overt circadian rhythmicity in P. confluens. To investigate whether a molecular clock is present, we analyzed frq transcription in constant darkness, and found circadian oscillation of frq with a peak in the subjective morning. This rhythm was temperature compensated. To identify additional clock-controlled genes, we performed RNA sequencing of two time points (subjective morning and evening). Circadian expression of two morning-specific genes was verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) over a full time course, whereas expression of two putative morning-specific and five putative evening-specific genes could not be verified as circadian. frq expression was synchronized, but not entrained by light. In summary, we have found evidence for two of the three main properties of circadian rhythms (free-running rhythm, temperature compensation) in P. confluens, suggesting that a circadian clock with rhythmically expressed frq is present in this basal filamentous ascomycete.