The evolution of complex multicellularity has been one of the major transitions in the history of life. In contrast to simple multicellular aggregates of cells, it has evolved only in a handful of lineages, including animals, embryophytes, red and brown algae, and fungi. Despite being a key step toward the evolution of complex organisms, the evolutionary origins and the genetic underpinnings of complex multicellularity are incompletely known. The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under laboratory conditions. We constructed a reference atlas of mushroom formation based on developmental transcriptome data of six species and comparisons of >200 whole genomes, to elucidate the core genetic program of complex multicellularity and fruiting body development in mushroom-forming fungi (Agaricomycetes). Nearly 300 conserved gene families and >70 functional groups contained developmentally regulated genes from five to six species, covering functions related to fungal cell wall remodeling, targeted protein degradation, signal transduction, adhesion, and small secreted proteins (including effector-like orphan genes). Several of these families, including F-box proteins, expansin-like proteins, protein kinases, and transcription factors, showed expansions in Agaricomycetes, many of which convergently expanded in multicellular plants and/or animals too, reflecting convergent solutions to genetic hurdles imposed by complex multicellularity among independently evolved lineages. This study provides an entry point to studying mushroom development and complex multicellularity in one of the largest clades of complex eukaryotic organisms. complex multicellularity | evolution | fungi | comparative genomics | fruiting body development F ungi represent a diverse lineage of complex multicellular organisms with a unique evolutionary history compared with complex multicellular animals, embryophytes, florideophytes, and laminarean brown algae (1-4). Within the fungal kingdom, complex multicellularity is discussed mostly in the context of fruiting bodies, which are found in at least eight independent lineages (2), of which the Pezizomycotina (Ascomycota) and the Agaricomycetes (Basidiomycota) contain the vast majority of species. The mushroom-forming fungi (Agaricomycetes) comprise >21,000 species and originated 350 million years ago (5), approximately coinciding with the origin of tetrapods. Fruiting bodies of mushroom-forming fungi have immense importance in agriculture, ecology, and medicine; they represent an important and sustainable food source, with favorable medicinal properties (e.g., antitumor, immunomodulatory) (6). Mushroom-forming fungi share a single origin of fruiting body formation that probably dates to the most recent common ancestor of the Agaricomycetes, Dacrymycetes, and Tremellomycetes (2).Fruiting body development in mushroom-forming fungi has been subject to surprisingly few studies (see, e.g., refs. 7-10), result...

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.

Antimicrobial peptides are promising alternative antimicrobial agents. However, little is known about whether resistance to small-molecule antibiotics leads to cross-resistance (decreased sensitivity) or collateral sensitivity (increased sensitivity) to antimicrobial peptides. We systematically addressed this question by studying the susceptibilities of a comprehensive set of 60 antibiotic-resistant Escherichia coli strains towards 24 antimicrobial peptides. Strikingly, antibiotic-resistant bacteria show a high frequency of collateral sensitivity to antimicrobial peptides, whereas cross-resistance is relatively rare. We identify clinically relevant multidrug-resistance mutations that increase bacterial sensitivity to antimicrobial peptides. Collateral sensitivity in multidrug-resistant bacteria arises partly through regulatory changes shaping the lipopolysaccharide composition of the bacterial outer membrane. These advances allow the identification of antimicrobial peptide-antibiotic combinations that enhance antibiotic activity against multidrug-resistant bacteria and slow down de novo evolution of resistance. In particular, when co-administered as an adjuvant, the antimicrobial peptide glycine-leucine-amide caused up to 30-fold decrease in the antibiotic resistance level of resistant bacteria. Our work provides guidelines for the development of efficient peptide-based therapies of antibiotic-resistant infections.

Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.genome engineering | synthetic biology | recombineering | off-target effects | methyl-directed mismatch repair

T he genus Armillaria causes root rot disease in both gymnoand angiosperms, in forests, parks, and even vineyards in more than 500 host plant species 1 across the world. Most Armillaria species are facultative necrotrophs, which, after colonizing and killing the root cambium, transition to a saprobic phase, decomposing dead woody tissues of the host. As saprotrophs, Armillaria spp. are white rot (WR) fungi, which can efficiently decompose all components of plant cell walls, including lignin, (hemi-)cellulose and pectin 2 . They produce fleshy fruiting bodies (honey mushrooms) that appear in large clumps around infected plants and produce sexual spores. The vegetative phase of Armillaria is predominantly diploid rather than dikaryotic like most basidiomycetes.Individuals of Armillaria can reach immense sizes and include the 'humongous fungus' , one of the largest terrestrial organisms on Earth 3 , measuring up to 965 hectares and 600 tons 4 , and can display a mutation rate ≅ 3 orders of magnitude lower than most filamentous fungi 5 . Individuals reach this immense size via growing rhizomorphs, dark mycelial strings 1-4 mm wide that allow the fungus to bridge gaps between food sources or host plants 1,6 (hence the name shoestring root rot). Rhizomorphs develop through the aggregation and coordinated parallel growth of hyphae, similar to some fruiting body tissues 7,8 . As migratory and exploratory organs, rhizomorphs can grow approximately 1 m yr −1 and cross several metres underground in search for new hosts, although roles in uptake and longrange translocation of nutrients have also been proposed 1,9,10 . Root contact by rhizomorphs is the main mode of infection by the fungus, which makes the prevention of recurrent infection in Armillariacontaminated areas particularly difficult 1 . Despite their huge impact on forestry, horticulture and agriculture, the genetics of the pathogenicity of Armillaria species is poorly understood. The only -omics data published so far have highlighted a substantial repertoire of plant cell wall degrading enzymes (PCWDE) and secreted proteins, among others, in A. mellea and A. solidipes 11,12 , while analyses of the genomes of other pathogenic basidiomycetes (such as Moniliophthora 13,14 , Heterobasidion 15 and Rhizoctonia 16 ) identified genes coding for PCWDEs, secreted and effector proteins or secondary metabolism (SM) as putative pathogenicity factors. However, the lifecycle and unique dispersal strategy of Armillaria prefigure other evolutionary routes to pathogenicity, which, along with other potential genomic factors (such as transposable elements 17 ) are not yet known.Here, we investigate genome evolution and the origin of pathogenicity in Armillaria using comparative genomics, transcriptomics

Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts during normal development contributes also to melanoma progression and metastasis. Although several epithelial-tomesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with reduced patient survival. Melanocytes are specialized cells in the skin that produce melanin, a pigment that is responsible for skin and hair color and that provides protection against ultraviolet (UV) radiation. During mouse embryogenesis, melanoblasts originate from the neural crest and migrate along a dorsolateral pathway from the neural tube to the developing dermis.1 Around embryonic day (E) E11 they move into the epidermis and eventually populate the developing hair follicle.2 Here they separate into two distinct populations: the differentiated pigmented melanocytes, which reside in the hair matrix, and the non-pigmented melanocyte stem cells (MSC) in the bulge. The latter cells are responsible for replenishing the hair follicle with new melanocytes during each hair cycle. Genetic studies in mice demonstrated the importance of several key players (such as sex-determining region Y (SRY)-box 10 (Sox10), paired-box 3 (Pax3), microphthalmia-associated transcription factor (Mitf), endothelin 3/endothelin receptor B (Edn3/ Ednrb), Kitl/Kit, Slug, cellular myelocytomatosis oncogene cellular homolog (cMyc) and b-Catenin (b-Cat)) for melanoblast cell fate specification, proliferation, migration and survival.2-4 The master regulator of the melanocyte development is MITF, which is spatio-temporally controlled by several key transcription factors such as SOX10, PAX3 and b-catenin.5-7 Fundamentally, MITF induces gene expression patterns that prompt a melanocyte to differentiate and initiate pigment production by activating genes important for melanin biosynthesis (such as Tyrosinase (Tyr), Dopachrome tautomerase (Dct), Tyrosinase-related protein 1 (Tyrp1) and

In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In , this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.

SignificanceAntibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. By building on multiplex automated genome engineering, we developed a method that enables precise mutagenesis of multiple, long genomic segments in multiple species without off-target modifications. Thereby, it enables the exploration of vast numbers of combinatorial genetic alterations in their native genomic context. This method is especially well-suited to screen the resistance profiles of antibiotic compounds. It allowed us to predict the evolution of resistance against antibiotics currently in clinical trials. We anticipate that it will be a useful tool to identify resistance-proof antibiotics at an early stage of drug development.