Next-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio-generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism's biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes.

The gene composition of present-day genomes has been shaped by a complicated evolutionary history, resulting in diverse distributions of genes across genomes. The pattern of presence and absence of a gene in different genomes is called its phylogenetic profile. It has been shown that proteins whose encoding genes have highly similar profiles tend to be functionally related: As these genes were gained and lost together, their encoded proteins can probably only perform their full function if both are present. However, a large proportion of genes encoding interacting proteins do not have matching profiles. In this study, we analysed one possible reason for this, namely that phylogenetic profiles can be affected by multi-functional proteins such as shared subunits of two or more protein complexes. We found that by considering triplets of proteins, of which one protein is multi-functional, a large fraction of disturbed co-occurrence patterns can be explained.

Fungi are renowned producers of natural compounds, also known as secondary metabolites (SMs) that display a wide array of biological activities. Typically, the genes that are involved in the biosynthesis of SMs are located in close proximity to each other in so-called secondary metabolite clusters. Many plant-pathogenic fungi secrete SMs during infection in order to promote disease establishment, for instance as cytocoxic compounds. Verticillium dahliae is a notorious plant pathogen that can infect over 200 host plants worldwide. However, the SM repertoire of this vascular pathogen remains mostly uncharted. To unravel the potential of V. dahliae to produce SMs, we performed in silico predictions and in-depth analyses of its secondary metabolite clusters. Using distinctive traits of gene clusters and the conserved signatures of core genes 25 potential SM gene clusters were identified. Subsequently, phylogenetic and comparative genomics analyses were performed, revealing that two putative siderophores, ferricrocin and TAFC, DHN-melanin and fujikurin may belong to the SM repertoire of V. dahliae.

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial coinhabitants. Also, plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne, fungal, xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life stage-specific manner and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota.

Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 (KMT6) and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for methylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted MS analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi.

The Arabidopsis thaliana Receptor-Like Protein RLP30 contributes to immunity against the fungal pathogen Sclerotinia sclerotiorum. Here we identify the RLP30-ligand as a small cysteine-rich protein (SCP) that occurs in many fungi and oomycetes and is also recognized by the Nicotiana benthamiana RLP RE02. However, RLP30 and RE02 share little sequence similarity and respond to different parts of the native/folded protein. Moreover, some Brassicaceae other than Arabidopsis also respond to a linear SCP peptide instead of the folded protein, suggesting that SCP is an eminent immune target that led to the convergent evolution of distinct immune receptors in plants. Surprisingly, RLP30 shows a second ligand specificity for a SCP-nonhomologous protein secreted by bacterial Pseudomonads. RLP30 expression in N. tabacum results in quantitatively lower susceptibility to bacterial, fungal and oomycete pathogens, thus demonstrating that detection of immunogenic patterns by Arabidopsis RLP30 is involved in defense against pathogens from three microbial kingdoms.

Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited availability of genomic resources for R. necatrix has complicated a thorough understanding of its infection biology. Here, we sequenced nine R. necatrix strains with Oxford Nanopore sequencing technology, and with DNA proximity ligation we generated a gapless assembly of one of the genomes into ten chromosomes. Whereas many filamentous pathogens display a so-called two-speed genome with more dynamic and more conserved compartments, the R. necatrix genome does not display such genome compartmentalization. It has recently been proposed that fungal plant pathogens may employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection. In the predicted secretome of R. necatrix, 26 putative antimicrobial effector proteins were identified, nine of which are expressed during plant colonization. Two of the candidates were tested, both of which were found to possess selective antimicrobial activity. Intriguingly, some of the inhibited bacteria are antagonists of R. necatrix growth in vitro and can alleviate R. necatrix infection on cotton plants. Collectively, our data show that R. necatrix encodes antimicrobials that are expressed during host colonization and that may contribute to modulation of host-associated microbiota to stimulate disease development.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

The taxonomic class of oomycetes contains numerous pathogens of plants and animals but is related to nonpathogenic diatoms and brown algae. Oomycetes have flexible genomes comprising large gene families that play roles in pathogenicity. The evolutionary processes that shaped the gene content have not yet been studied by applying systematic tree reconciliation of the phylome of these species. We analyzed evolutionary dynamics of ten Stramenopiles. Gene gains, duplications, and losses were inferred by tree reconciliation of 18,459 gene trees constituting the phylome with a highly supported species phylogeny. We reconstructed a strikingly large last common ancestor of the Stramenopiles that contained ~10,000 genes. Throughout evolution, the genomes of pathogenic oomycetes have constantly gained and lost genes, though gene gains through duplications outnumber the losses. The branch leading to the plant pathogenic Phytophthora genus was identified as a major transition point characterized by increased frequency of duplication events that has likely driven the speciation within this genus. Large gene families encoding different classes of enzymes associated with pathogenicity such as glycoside hydrolases are formed by complex and distinct patterns of duplications and losses leading to their expansion in extant oomycetes. This study unveils the large-scale evolutionary dynamics that shaped the genomes of pathogenic oomycetes. By the application of phylogenetic based analyses methods, it provides additional insights that shed light on the complex history of oomycete genome evolution and the emergence of large gene families characteristic for this important class of pathogens.

Proteins play a key role in cellular life. They do not act alone but are organised in complexes. Throughout the life of a cell, complexes are dynamic in their composition due to attachments and shared components. Experimental and computational evidence indicate that consecutive addition and secondary losses of components played a major role in the evolution of some complexes, mostly without affecting the core function. Here, we analysed in a large scale approach whether this flexibility in evolution is only limited to a distinct number of complexes or represents a more general trend.

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca(2+) ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Banana is the most popular and most exported fruit and also a major food crop for millions of people around the world. Despite its importance and the presence of serious disease threats, research into this crop is limited. One of those is Panama disease or Fusarium wilt. In the previous century Fusarium wilt wiped out the "Gros Michel" based banana industry in Central America. The epidemic was eventually quenched by planting "Cavendish" bananas. However, 50 years ago the disease recurred, but now on "Cavendish" bananas. Since then the disease has spread across South-East Asia, to the Middle-East and the Indian subcontinent and leaped into Africa. Here, we report the presence of Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4) in "Cavendish" plantations in Laos, Myanmar, and Vietnam. A combination of classical morphology, DNA sequencing, and phenotyping assays revealed a very close relationship between the Foc TR4 strains in the entire Greater Mekong Subregion (GMS), which is increasingly prone to intensive banana production. Analyses of single-nucleotide polymorphisms enabled us to initiate a phylogeography of Foc TR4 across three geographical areas-GMS, Indian subcontinent, and the Middle East revealing three distinct Foc TR4 sub-lineages. Collectively, our data place these new incursions in a broader agroecological context and underscore the need for awareness campaigns and the implementation of validated quarantine measures to prevent further international dissemination of Foc TR4.

Pseudocercospora fijiensis is the causal agent of the black leaf streak disease (BLSD) of banana. Bananas are important global export commodities and a major staple food. Their susceptibility to BLSD pushes disease management towards excessive fungicide use, largely relying on multisite inhibitors and sterol demethylation inhibitors (DMIs). These fungicides are ubiquitous in plant disease control, targeting the CYP51 enzyme. We examined sensitivity to DMIs in P. fijiensis field isolates collected from various major banana production zones in Colombia, Costa Rica, Dominican Republic, Ecuador, the Philippines, Guadalupe, Martinique and Cameroon and determined the underlying genetic reasons for the observed phenotypes.

Genomes store information at scales beyond the linear nucleotide sequence, which impacts genome function at the level of an individual, while influences on populations and long-term genome function remains unclear. Here, we addressed how physical and chemical DNA characteristics influence genome evolution in the plant pathogenic fungus Verticillium dahliae. We identified incomplete DNA methylation of repetitive elements, associated with specific genomic compartments originally defined as Lineage-Specific (LS) regions that contain genes involved in host adaptation. Further chromatin characterization revealed associations with features such as H3 Lys-27 methylated histones (H3K27me3) and accessible DNA. Machine learning trained on chromatin data identified twice as much LS DNA as previously recognized, which was validated through orthogonal analysis, and we propose to refer to this DNA as adaptive genomic regions. Our results provide evidence that specific chromatin profiles define adaptive genomic regions, and highlight how different epigenetic factors contribute to the organization of these regions.

Sensing external signals and transducing these into intracellular responses requires a molecular signaling system that is crucial for every living organism. Two important eukaryotic signal transduction pathways that are often interlinked are G-protein signaling and phospholipid signaling. Heterotrimeric G-protein subunits activated by G-protein-coupled receptors (GPCRs) are typical stimulators of phospholipid signaling enzymes such as phosphatidylinositol phosphate kinases (PIPKs) or phospholipase C (PLC). However, a direct connection between the two pathways likely exists in oomycetes and slime molds, as they possess a unique class of GPCRs that have a PIPK as an accessory domain. In principle, these so-called GPCR-PIPKs have the capacity of perceiving an external signal (via the GPCR domain) that, via PIPK, directly activates downstream phospholipid signaling. Here we reveal the sporadic occurrence of GPCR-PIPKs in all eukaryotic supergroups, except for plants. Notably, all species having GPCR-PIPKs are unicellular microorganisms that favor aquatic environments. Phylogenetic analysis revealed that GPCR-PIPKs are likely ancestral to eukaryotes and significantly expanded in the last common ancestor of oomycetes. In addition to GPCR-PIPKs, we identified five hitherto-unknown classes of GPCRs with accessory domains, four of which are universal players in signal transduction. Similarly to GPCR-PIPKs, this enables a direct coupling between extracellular sensing and downstream signaling. Overall, our findings point to an ancestral signaling system in eukaryotes where GPCR-mediated sensing is directly linked to downstream responses.IMPORTANCE G-protein-coupled receptors (GPCRs) are central sensors that activate eukaryotic signaling and are the primary targets of human drugs. In this report, we provide evidence for the widespread though limited presence of a novel class of GPCRs in a variety of unicellular eukaryotes. These include free-living organisms and organisms that are pathogenic for plants, animals, and humans. The novel GPCRs have a C-terminal phospholipid kinase domain, pointing to a direct link between sensing external signals via GPCRs and downstream intracellular phospholipid signaling. Genes encoding these receptors were likely present in the last common eukaryotic ancestor and were lost during the evolution of higher eukaryotes. We further describe five other types of GPCRs with a catalytic accessory domain, the so-called GPCR-bigrams, four of which may potentially have a role in signaling. These findings shed new light onto signal transduction in microorganisms and provide evidence for alternative eukaryotic signaling pathways.

Chitin-binding lysin motif (LysM) effectors contribute to the virulence of various plant-pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil-borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage-specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage-specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin-induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage-specific LysM effector of strain VdLs17 contributes to virulence in planta.

Plant infection by oomycete pathogens is a complex process. It requires precise expression of a plethora of genes in the pathogen that contribute to a successful interaction with the host. Whereas much effort has been made to uncover the molecular systems underlying this infection process, mechanisms of transcriptional regulation of the genes involved remain largely unknown. We performed the first systematic de-novo DNA motif discovery analysis in Phytophthora. To this end, we utilized the genome sequence of the late blight pathogen Phytophthora infestans and two related Phytophthora species (P. ramorum and P. sojae), as well as genome-wide in planta gene expression data to systematically predict 19 conserved DNA motifs. This catalog describes common eukaryotic promoter elements whose functionality is supported by the presence of orthologs of known general transcription factors. Together with strong functional enrichment of the common promoter elements towards effector genes involved in pathogenicity, we obtained a new and expanded picture of the promoter structure in P. infestans. More intriguingly, we identified specific DNA motifs that are either highly abundant or whose presence is significantly correlated with gene expression levels during infection. Several of these motifs are observed upstream of genes encoding transporters, RXLR effectors, but also transcriptional regulators. Motifs that are observed upstream of known pathogenicity-related genes are potentially important binding sites for transcription factors. Our analyses add substantial knowledge to the as of yet virtually unexplored question regarding general and specific gene regulation in this important class of pathogens. We propose hypotheses on the effects of cis-regulatory motifs on the gene regulation of pathogenicity-related genes and pinpoint motifs that are prime targets for further experimental validation.

The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications, with a major contribution from sterol demethylation-inhibitors (DMIs). The continued use of DMIs places considerable selection pressure on natural P. fijiensis populations, enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non-synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α-demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19-bp element in the wild-type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A polymerase chain reaction (PCR) assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wt promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional up-regulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain, as well as promoter inserts, contribute to the reduced DMI sensitivity of P. fijiensis. These results provide new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.

Genome-scale metabolic models (GEMs) provide a functional view of the complex network of biochemical reactions in the living cell. Initially mainly applied to reconstruct the metabolism of model organisms, the availability of increasingly sophisticated reconstruction methods and more extensive biochemical databases now make it possible to reconstruct GEMs for less well-characterized organisms, and have the potential to unravel the metabolism in pathogen-host systems. Here, we present a GEM for the oomycete plant pathogen Phytophthora infestans as a first step towards an integrative model with its host. We predict the biochemical reactions in different cellular compartments and investigate the gene-protein-reaction associations in this model to obtain an impression of the biochemical capabilities of P. infestans. Furthermore, we generate life stage-specific models to place the transcriptomic changes of the genes encoding metabolic enzymes into a functional context. In sporangia and zoospores, there is an overall down-regulation, most strikingly reflected in the fatty acid biosynthesis pathway. To investigate the robustness of the GEM, we simulate gene deletions to predict which enzymes are essential for in vitro growth. This model is an essential first step towards an understanding of P. infestans and its interactions with plants as a system, which will help to formulate new hypotheses on infection mechanisms and disease prevention.