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Plant pathogens secrete effector molecules during host invasion to promote colonization. However, some of these effectors become recognized by host receptors to mount a defence response and establish immunity. Recently, a novel resistance was identified in wild tomato, mediated by the single dominant V2 locus, to control strains of the soil-borne vascular wilt fungus Verticillium dahliae that belong to race 2. With comparative genomics of race 2 strains and resistance-breaking race 3 strains, we identified the avirulence effector that activates V2 resistance, termed Av2. We identified 277 kb of race 2-specific sequence comprising only two genes encoding predicted secreted proteins that are expressed during tomato colonization. Subsequent functional analysis based on genetic complementation into race 3 isolates and targeted deletion from the race 1 isolate JR2 and race 2 isolate TO22 confirmed that one of the two candidates encodes the avirulence effector Av2 that is recognized in V2 tomato plants. Two Av2 allelic variants were identified that encode Av2 variants that differ by a single acid. Thus far, a role in virulence could not be demonstrated for either of the two variants.

Transposable elements (TEs) are a major source of genetic and regulatory variation in their host genome and are consequently thought to play important roles in evolution. Many fungal and oomycete plant pathogens have evolved dynamic and TE-rich genomic regions containing genes that are implicated in host colonization. TEs embedded in these regions have typically been thought to accelerate the evolution of these genomic compartments, but little is known about their dynamics in strains that harbor them. Here, we used whole-genome sequencing data of 42 strains of the fungal plant pathogen Verticillium dahliae to systematically identify polymorphic TEs that may be implicated in genomic as well as in gene expression variation. We identified 2,523 TE polymorphisms and characterize a subset of 8% of the TEs as dynamic elements that are evolutionary younger, less methylated, and more highly expressed when compared with the remaining 92% of the TE complement. As expected, the dynamic TEs are enriched in the dynamic genomic regions. Besides, we observed an association of dynamic TEs with pathogenicity-related genes that localize nearby and that display high expression levels. Collectively, our analyses demonstrate that TE dynamics in V. dahliae contributes to genomic variation, correlates with expression of pathogenicity-related genes, and potentially impacts the evolution of dynamic genomic regions.Significance statementTransposable elements (TEs) are ubiquitous components of genomes and are major sources of genetic and regulatory variation. Many plant pathogens have evolved TE-rich genomic regions containing genes with roles in host colonization, and TEs are thought to contribute to accelerated evolution of these dynamic regions. We analyzed the fungal plant pathogen Verticillium dahliae to identify TE variation between strains and to demonstrate that polymorphic TEs have specific characteristic that separates them from the majority of TEs. Polymorphic TEs are enriched in dynamic genomic regions and are associated with structural variants and highly expressed pathogenicity-related genes. Collectively, our results provide evidence for the hypothesis that dynamic TEs contribute to increased genomic diversity, functional variation, and the evolution of dynamic genomic regions.

Differential growth conditions typically trigger global transcriptional responses in filamentous fungi. Such fungal responses to environmental cues involve epigenetic regulation, including chemical histone modifications. It has been proposed that conditionally expressed genes, such as those that encode secondary metabolites but also effectors in pathogenic species, are often associated with a specific histone modification, lysine27 methylation of H3 (H3K27me3). However, thus far no analyses on the global H3K27me3 profiles have been reported under differential growth conditions in order to assess if H3K27me3 dynamics governs differential transcriptional. Using ChIP- and RNA-sequencing data from the plant pathogenic fungus Verticillium dahliae grown on three in vitro cultivation media, we now show that a substantial number of the identified H3K27me3 domains globally display stable profiles among these growth conditions. However, we do observe local quantitative differences in H3K27me3 ChIP-seq signal that associate with differentially transcribed genes between media. Comparing the in vitro results to expression during plant infection suggests that in planta induced genes require chromatin remodelling to achieve expression. Overall, our results demonstrate that although some loci display H3K27me3 dynamics that can contribute to transcriptional variation, other loci do not show such dependence. Thus, we conclude that while H3K27me3 is required for transcriptional repression, it is not a conditionally responsive global regulator of differential transcription. We propose that the H3K27me3 domains that do not undergo dynamic methylation may contribute to transcription through other mechanisms or may serve additional genomic regulatory functions. IMPORTANCE In many organisms, including filamentous fungi, epigenetic mechanisms that involve chemical and physical modifications of DNA without changing the genetic sequence have been implicated in transcriptional responses upon developmental or environmental cues. In fungi, facultative heterochromatin that can de-condense to allow transcription in response to developmental changes or to environmental stimuli is characterized by tri-methylation of lysine 27 on histone H3 (H3K27me3), and H3K27me3 has been implicated in transcriptional regulation although precise mechanisms and functions remain enigmatic. Based on ChIP- and RNA-sequencing data, we show for the soil-borne broad host range vascular wilt plant pathogenic fungus Verticillium dahliae that although some loci display H3K27me3 dynamics that can contribute to transcriptional variation, other loci do not show such dependence. Thus, although we recognize that H3K27me3 is required for transcriptional repression, we also conclude that this mark is not a conditionally responsive global regulator of differential transcription upon responses to environmental cues.

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.

DNA methylation is an important epigenetic control mechanism that in many fungi is restricted to genomic regions containing transposons. Two DNA methyltransferases, Dim2 and Dnmt5, are known to perform methylation at cytosines in fungi. While most ascomycete fungi encode both Dim2 and Dnmt5, only few functional studies have been performed in species containing both. In this study, we report functional analysis of bothDim2andDnmt5in the plant pathogenic fungusVerticillium dahliae. Our results show that Dim2, but not Dnmt5 or the putative sexual-cycle related DNA methyltransferase Rid, is responsible for nearly all DNA methylation. Single or double DNA methyltransferase mutants did not show altered development, virulence, or transcription of genes or transposons. In contrast,Hp1andDim5mutants that are impacted in chromatin-associated processes upstream of DNA methylation are severely affected in development and virulence and display extensive transcriptional reprogramming in specific hypervariable genomic regions (so-called lineage-specific (LS) regions) that contain genes associated with host colonization. As these LS regions are largely devoid of DNA methylation and of Hp1- and Dim5-associated heterochromatin, the differential transcription is likely caused by pleiotropic effects rather than by differential DNA methylation. Overall, our study suggests that Dim2 is the main DNA methyltransferase inV. dahliaeand, in conjunction with work on other fungi, is likely the main active DNMT in ascomycetes, irrespective ofDnmt5presence. We speculate that Dnmt5 acts under specific, presently enigmatic, conditions or, alternatively, acts in DNA-associated processes other than DNA methylation.

SUMMARYPlant pathogens secrete effector molecules during host invasion to promote host colonization. However, some of these effectors become recognized by host receptors, encoded by resistance genes, to mount defense response and establish immunity. Recently, a novel resistance was identified in tomato, mediated by the single dominant V2 locus, to control strains of the soil-borne vascular wilt fungus Verticillium dahliae that belong to race 2. We performed comparative genomics between race 2 strains and resistance-breaking race 3 strains to identify the avirulence effector that activates V2 resistance, termed Av2. We identified 277 kb of race 2-specific sequence comprising only two genes that encode predicted secreted proteins, both of which are expressed by V. dahliae during tomato colonization. Subsequent functional analysis based on genetic complementation into race 3 isolates confirmed that one of the two candidates encodes the avirulence effector Av2 that is recognized in V2 tomato plants. The identification of Av2 will not only be helpful to select tomato cultivars that are resistant to race 2 strains of V. dahliae, as the corresponding V2 resistance gene has not yet been mapped, but also to monitor adaptations in the V. dahliae population to deployment of V2-containing tomato cultivars in agriculture.

Understanding the complex information stored in a genome remains challenging since multiple connected regulatory mechanisms act at various scales to determine function. Increased comprehension of genome function at scales beyond contiguous nucleotides will help understand genetic diseases, the emergence of pathogenesis, and more broadly the genomics of adaptation. Here we report the analysis of DNA methylation, histone modification, and DNA accessibility in the plant pathogenic vascular wilt fungusVerticillium dahliae. Functional analysis details that DNA methylation is restricted to repetitive elements, such as transposable element DNA, but interestingly only some repetitive DNA is methylated. This incomplete DNA methylation is associated with repetitive DNA residing in specific compartments of the genome that were previously defined as Lineage-Specific (LS) regions. These regions are hypervariable betweenV. dahliaeisolates and contain genes that support host colonization and adaptive traits. LS regions are associated with H3 Lys-27 methylated histones (H3K27me3), and repetitive DNA within LS regions are more transcriptionally active and have increased DNA accessibility, representing a hybrid chromatin state when compared to repetitive regions within the core genome. We used machine learning algorithms trained on epigenetic and DNA accessibility data to predict LS regions with high recall, identifying approximately twice as much LS DNA in theV. dahliaegenome as previously recognized. Collectively, these results characterize LS regions in an intermediate chromatin state and provide evidence that links chromatin features and genome architecture to adaptive regions within the genome.

The fungal genus Verticillium contains ten species, some of which are notorious plant pathogens causing vascular wilt diseases in host plants, while others are known as saprophytes and opportunistic plant pathogens. Whereas the genome of V. dahliae, the most notorious plant pathogen of the genus, has been well characterized, evolution and speciation of other members of the genus received little attention thus far. Here, we sequenced the genomes of the nine haploid Verticillium spp. to study evolutionary trajectories of their divergence from a last common ancestor. Frequent occurrence of chromosomal rearrangement and gene family loss was identified. In addition to ∼11 000 genes that are shared at least between two species, only 200-600 species-specific genes occur. Intriguingly, these species-specific genes show different features than the shared genes.

Transposable elements are powerful drivers of genome evolution in many eukaryotes. Although they are mostly considered as 'selfish' genetic elements, increasing evidence suggests that they contribute to genetic variability; particularly under stress conditions. Over the past few years, the role of transposable elements during host-microbe interactions has been recognised. It has been proposed that many pathogenic microbes have evolved a 'two-speed' genome with regions that show increased variability and that are enriched in transposable elements and pathogenicity-related genes. Plants similarly display structured genomes with transposable-element-rich regions that mediate accelerated evolution. Immune receptor genes typically reside in such regions. Various mechanisms have recently been identified through which transposable elements contribute to the coevolution between plants and their associated microbes. Transposable elements are powerful drivers of adaptive genome evolution in plants and in symbiotic microbes, and contribute to their coevolution.Analysis of large next-generation sequencing datasets fuels a better understanding of the precise role of transposable elements in genome evolution, revealing active as well as passive contributions.Transposable elements contribute to genome evolution through diverse mechanisms, by mediating structural variations, gene inactivation, gene copy variation, but also by affecting gene expression.Further mechanistic understanding of the role of transposable elements in genome evolution will come from detailed structural analysis of chromatin.

Plant pathogens continuously evolve to evade host immune responses. During host colonization, many fungal pathogens secrete effectors to perturb such responses, but these in turn may become recognized by host immune receptors. To facilitate the evolution of effector repertoires, such as the elimination of recognized effectors, effector genes often reside in genomic regions that display increased plasticity, a phenomenon that is captured in the two‐speed genome hypothesis. The genome of the vascular wilt fungus Verticillium dahliae displays regions with extensive presence/absence polymorphisms, so‐called lineage‐specific regions, that are enriched in in planta‐induced putative effector genes. As expected, comparative genomics reveals differential degrees of sequence divergence between lineage‐specific regions and the core genome. Unanticipated, lineage‐specific regions display markedly higher sequence conservation in coding as well as noncoding regions than the core genome. We provide evidence that disqualifies horizontal transfer to explain the observed sequence conservation and conclude that sequence divergence occurs at a slower pace in lineage‐specific regions of the V. dahliae genome. We hypothesize that differences in chromatin organisation may explain lower nucleotide substitution rates in the plastic, lineage‐specific regions of V. dahliae.

Along withPlasmopara destructor, Peronosopora belbahriihas arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was ca. 35.4 Mbp in length, with an N50 scaffold length of ca. 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of ca. 40.1 kbp. From the repeat-masked genome 9049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16 % of the genome consists of repetitive sequences, and based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies ofPe. belbahrii. Peronospora belbahriihas undergone a high degree of convergent evolution, reflecting its obligate biotrophic lifestyle. Features of its secretome, signalling networks, and promoters are presented, and some patterns are hypothesised to reflect the high degree of host specificity inPeronosporaspecies. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP) have an extremely broad taxonomic distribution; they occur in bacteria, fungi, and oomycetes. NLPs come in two forms, those that are cytotoxic to eudicot plants and those that are noncytotoxic. Cytotoxic NLPs bind to glycosyl inositol phosphoryl ceramide (GIPC) sphingolipids that are abundant in the outer leaflet of plant plasma membranes. Binding allows the NLP to become cytolytic in eudicots but not monocots. The function of noncytotoxic NLPs remains enigmatic, but the expansion of NLP genes in oomycete genomes suggests they are important. Several plant species have evolved the capacity to recognize NLPs as molecular patterns and trigger plant immunity, e.g., Arabidopsis thaliana detects nlp peptides via the receptor-like protein RLP23. In this review, we provide a historical perspective from discovery to understanding of molecular mechanisms and describe the latest developments in the NLP field to shed light on these fascinating microbial proteins.

Horizontal gene transfer (HGT) involves the transmission of genetic material between distinct evolutionary lineages and can be an important source of biological innovation. Reports of inter-kingdom HGT to eukaryotic microbial pathogens have accumulated over recent years. Verticillium dahliae is a notorious plant pathogen that causes vascular wilt disease on hundreds of plant species, resulting in high economic losses every year. Previously, the effector gene Ave1 and a glucosyltransferase-encoding gene were identified as virulence factor-encoding genes that were proposed to be horizontally acquired from a plant and a bacterial donor, respectively. However, to what extent HGT contributed to the overall genome composition of V. dahliae remained elusive. Here, we systematically searched for evidence of inter-kingdom HGT events in the genome of V. dahliae and provide evidence for extensive horizontal gene acquisition from bacterial origin.

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near-finished genome sequence for B. cinerea strain B05.10 is reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.

Microbial pathogens cause devastating diseases on economically and ecologically important plant species, threatening global food security, and causing billions of dollars of losses annually. During the infection process, pathogens secrete so-called effectors that support host colonization, often by deregulating host immune responses. Over the last decades, much of the research on molecular plant-microbe interactions has focused on the identification and functional characterization of such effectors. The increasing availability of sequenced plant pathogen genomes has enabled genomics-based discovery of effector candidates. Nevertheless, identification of full plant pathogen effector repertoires is often hampered by erroneous gene annotation and the localization effector genes in genomic regions that are notoriously difficult to assemble. Here, we argue that recent advances in genome sequencing technologies, genome assembly, gene annotation, as well as effector identification methods hold promise to disclose complete and correct effector repertoires. This allows to exploit complete effector repertoires, and knowledge of their diversity within pathogen populations, to develop durable and sustainable resistance breeding strategies, disease control, and management of plant pathogens.

Contains fulltext : 168245.pdf (Publisher’s version ) (Open Access) Genomic plasticity enables adaptation to changing environments, which is especially relevant for pathogens that engage in "arms races" with their hosts. In many pathogens, genes mediating virulence cluster in highly variable, transposon-rich, physically distinct genomic compartments. However, understanding of the evolution of these compartments, and the role of transposons therein, remains limited. Here, we show that transposons are the major driving force for adaptive genome evolution in the fungal plant pathogen Verticillium dahliae We show that highly variable lineage-specific (LS) regions evolved by genomic rearrangements that are mediated by erroneous double-strand repair, often utilizing transposons. We furthermore show that recent genetic duplications are enhanced in LS regions, against an older episode of duplication events. Finally, LS regions are enriched in active transposons, which contribute to local genome plasticity. Thus, we provide evidence for genome shaping by transposons, both in an active and passive manner, which impacts the evolution of pathogen virulence.

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., In total, 25 potential SMCs were identified in the genome of the plant pathogenic fungus Verticillium dahliae, including loci that can be implicated in DHN-melanin, ferricrocin, triacetyl fusarinine and fujikurin production.

Hybridization is an important evolutionary mechanism that can enable organisms to adapt to environmental challenges. It has previously been shown that the fungal allodiploid species Verticillium longisporum, causal agent of Verticillium stem striping in rape seed, has originated from at least three independent hybridization events between two haploid Verticillium species. To reveal the impact of genome duplication as a consequence of the hybridization, we studied the genome and transcriptome dynamics upon two independent V. longisporum hybridization events, represented by the hybrid lineages “A1/D1” and “A1/D3”. We show that the V. longisporum genomes are characterized by extensive chromosomal rearrangements, including between parental chromosomal sets. V. longisporum hybrids display signs of evolutionary dynamics that are typically associated with the aftermath of allodiploidization, such as haploidization and a more relaxed gene evolution. Expression patterns of the two sub-genomes within the two hybrid lineages are more similar than those of the shared A1 parent between the two lineages, showing that expression patterns of the parental genomes homogenized within a lineage. However, as genes that display differential parental expression in planta do not typically display the same pattern in vitro, we conclude that sub-genome-specific responses occur in both lineages. Overall, our study uncovers the genomic and transcriptomic plasticity during evolution of the filamentous fungal hybrid V. longisporum and illustrate its adaptive potential.ImportanceVerticillium is a genus of plant-associated fungi that include a handful of plant pathogens that collectively affect a wide range of hosts. On several occasions, haploid Verticillium species hybridized into the stable allodiploid species Verticillium longisporum, which is, in contrast to haploid Verticillium species, a Brassicaceae specialist. Here, we studied the evolutionary genome and transcriptome dynamics of V. longisporum and the impact of the hybridization. V. longisporum genomes display a mosaic structure due do genomic rearrangements between the parental chromosome sets. Similar to other allopolyploid hybrids, V. longisporum displays an ongoing loss of heterozygosity and a more relaxed gene evolution. Also, differential parental gene expression is observed, with an enrichment for genes that encode secreted proteins. Intriguingly, the majority of these genes displays sub-genome-specific responses under differential growth conditions. In conclusion, hybridization has incited the genomic and transcriptomic plasticity that enables adaptation to environmental changes in a parental allele-specific fashion.

Selection pressure impacts genomes unevenly, as different genes adapt with differential speed to establish an organism’s optimal fitness. Plant pathogens co-evolve with their hosts, which implies continuously adaption to evade host immunity. Effectors are secreted proteins that mediate immunity evasion, but may also typically become recognized by host immune receptors. To facilitate effector repertoire alterations, in many pathogens, effector genes reside in dynamic genomic regions that are thought to display accelerated evolution, a phenomenon that is captured by the two-speed genome hypothesis. The genome of the vascular wilt pathogen Verticillium dahliae has been proposed to obey to a similar two-speed regime with dynamic, lineage-specific regions that are characterized by genomic rearrangements, increased transposable element activity and enrichment in in planta-induced effector genes. However, little is known of the origin of, and sequence diversification within, these lineage-specific regions. Based on comparative genomics among Verticillium spp. we now show differential sequence divergence between core and lineage-specific genomic regions of V. dahliae. Surprisingly, we observed that lineage-specific regions display markedly increased sequence conservation. Since single nucleotide diversity is reduced in these regions, host adaptation seems to be merely achieved through presence/absence polymorphisms. Increased sequence conservation of genomic regions important for pathogenicity is an unprecedented finding for filamentous plant pathogens and signifies the diversity of genomic dynamics in host-pathogen co-evolution.

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.

The Alternaria genus consists of saprophytic fungi as well as plant-pathogenic species that have significant economic impact. To date, the genomes of multiple Alternaria species have been sequenced. These studies have yielded valuable data for molecular studies on Alternaria fungi. However, most of the current Alternaria genome assemblies are highly fragmented, thereby hampering the identification of genes that are involved in causing disease. Here, we report a gapless genome assembly of A. solani, the causal agent of early blight in tomato and potato. The genome assembly is a significant step toward a better understanding of pathogenicity of A. solani.

SUMMARYThe fungal genus Verticillium contains ten species, some of which are notorious plant pathogens causing vascular wilt diseases in host plants, while others are known as saprophytes and opportunistic plant pathogens. Whereas the genome of V. dahliae, the most notorious plan pathogen of the genus, has been well characterized, evolution and speciation of other members of the genus received little attention thus far. Here, we sequenced the genomes of the nine haploid Verticillium spp. to study evolutionary trajectories of their divergence from a last common ancestor. Frequent occurrence of chromosomal rearrangement and gene family loss was identified. In addition to ~11,000 core genes that are shared among all species, only 200-600 species-specific genes occur. Intriguingly, these species-specific genes show different features than core genes.

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 characterized organisms as well, 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 get an impression of the biochemical capabilities of P. infestans. Furthermore, we generate life stage-specific models to place the transcriptomic changes of genes encoding metabolic enzymes into a functional context. In sporangia and zoospores there is an overall downregulation, 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. While there is room for improvement, this first model is an essential 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.

A DNA modification - methylation of cytosines and adenines - has important roles in diverse processes such as regulation of gene expression and genome stability, yet until recently adenine methylation had been considered to be only a hallmark of prokaryotes. A new study identifies abundant adenine methylation of transcriptionally active genes in early-diverging fungi that, together with recent other work, emphasizes the importance of adenine methylation in eukaryotes.

Sclerotinia sclerotiorum is a phytopathogenic fungus with over 400 hosts including numerous economically important cultivated species. This contrasts many economically destructive pathogens that only exhibit a single or very few hosts. Many plant pathogens exhibit a "two-speed" genome. So described because their genomes contain alternating gene rich, repeat sparse and gene poor, repeat-rich regions. In fungi, the repeat-rich regions may be subjected to a process termed repeat-induced point mutation (RIP). Both repeat activity and RIP are thought to play a significant role in evolution of secreted virulence proteins, termed effectors. We present a complete genome sequence of S. sclerotiorum generated using Single Molecule Real-Time Sequencing technology with highly accurate annotations produced using an extensive RNA sequencing data set. We identified 70 effector candidates and have highlighted their in planta expression profiles. Furthermore, we characterized the genome architecture of S. sclerotiorum in comparison to plant pathogens that exhibit "two-speed" genomes. We show that there is a significant association between positions of secreted proteins and regions with a high RIP index in S. sclerotiorum but we did not detect a correlation between secreted protein proportion and GC content. Neither did we detect a negative correlation between CDS content and secreted protein proportion across the S. sclerotiorum genome. We conclude that S. sclerotiorum exhibits subtle signatures of enhanced mutation of secreted proteins in specific genomic compartments as a result of transposition and RIP activity. However, these signatures are not observable at the whole-genome scale.

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., Molecular Plant Pathology, 18 (4), ISSN:1464-6722, ISSN:1364-3703

Genomic plasticity enables adaptation to changing environments, which is especially relevant for pathogens that engage in arms races with their hosts. In many pathogens, genes mediating aggressiveness cluster in highly variable, transposon-rich, physically distinct genomic compartments. However, understanding of the evolution of these compartments, and the role of transposons therein, remains limited. We now show that transposons are the major driving force for adaptive genome evolution in the fungal plant pathogen Verticillium dahliae. Highly variable genomic regions evolved by frequent segmental duplications mediated by erroneous homologous recombination, often utilizing transposons, leading to genetic material that is free to diverge. Intriguingly, the duplicated regions are enriched in active transposons that further contribute to local genome plasticity. Thus, we provide evidence for genome shaping by transposons, both in an active and passive manner, which impacts the evolution of pathogen aggressiveness.

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions.

An organism’s adaptation to changing environments is fueled by its genetic variability, which is established by mechanisms ranging from single-nucleotide polymorphisms to large-scale structural variations, all of which affect chromosomal shape, organization, and gene content [1]. These processes are particularly relevant for pathogens that must respond to continual selection pressure arising from host immune systems that evolved to detect the presence or activity of potentialmicrobial pathogens through a variety of invasion patterns [2]. In their adaptive response, pathogens evolve strategies, often involving secreted effectormolecules, to overcome host immunity and support host colonization [3]. Thus, it can be anticipated that this coevolutionary arms race leads to highly specific interactions between adapted pathogens and their specific hosts. Paradoxically, particular pathogens successfully colonize a broad range of hosts, yet how such pathogens cope in arms races with such a diversity of hosts remains unknown.

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.

Vascular wilts caused by Verticillium spp. are destructive plant diseases affecting hundreds of hosts. Only a few Verticillium spp. are causal agents of vascular wilt diseases, of which V. dahliae is the most notorious pathogen, and several V. dahliae genomes are available. In contrast, V. tricorpus is mainly known as a saprophyte and causal agent of opportunistic infections. Based on a hybrid approach that combines second and third generation sequencing, a near-gapless V. tricorpus genome assembly was obtained. With comparative genomics, we sought to identify genomic features in V. dahliae that confer the ability to cause vascular wilt disease. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. Intriguingly, V. tricorpus contains significantly fewer repetitive elements and an extended spectrum of secreted carbohydrate- active enzymes when compared with V. dahliae. In conclusion, we highlight the technical advances of a hybrid sequencing and assembly approach and show that the saprophyte V. tricorpus shares many hallmark features with the pathogen V. dahliae.

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., Author Summary Every genome of current day species contains a very unique selection of genes. Why a specific genome is composed of exactly those genes is determined by many factors, but often not resolvable. It seems plausible that interacting genes would either occur together or be absent together, because if one of them is alone, it might not be able to perform its function properly, just as a bolt can only perform its function together with a nut and vice versa. However, it turns out that interacting genes very often do not nicely co-occur across a wide range of species, and frequently one gene can be found but the other not. In this study, we investigated the co-occurrences of multi-functional proteins and found that they are often maintained in a genome, even if one of their interaction partners is lost. This is because they can still perform some functions with other interaction partners that are still present. We can show that this has a noticeable effect on genome compositions and can explain otherwise surprisingly mismatching co-occurrence patterns of interacting genes.

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.

Biotrophic plant pathogens secrete effector proteins that are important for infection of the host. The aim of this study was to identify effectors of the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) that are expressed during infection of its natural host Arabidopsis thaliana. Infection-related transcripts were identified from Expressed Sequence Tags (ESTs) derived from leaves of the susceptible Arabidopsis Ws eds1-1 mutant inoculated with the highly virulent Hpa isolate Waco9. Assembly of 6364 ESTs yielded 3729 unigenes, of which 2164 were Hpa-derived. From the translated Hpa unigenes, 198 predicted secreted proteins were identified. Of these, 75 were found to be Hpa-specific and six isolate Waco9-specific. Among 42 putative effectors identified there were three Elicitin-like proteins, 16 Cysteine-rich proteins and 18 host-translocated RXLR effectors. Sequencing of alleles in different Hpa isolates revealed that five RXLR genes show signatures of diversifying selection. Thus, EST analysis of Hpa-infected Arabidopsis is proving to be a powerful method for identifying pathogen effector candidates expressed during infection. Delivery of the Waco9-specific protein RXLR29 in planta revealed that this effector can suppress PAMP-triggered immunity and enhance disease susceptibility. We propose that differences in host colonization can be conditioned by isolate-specific effectors.

Oomycetes comprise a diverse group of organisms that morphologically resemble fungi but belong to the stramenopile lineage within the supergroup of chromalveolates. Recent studies have shown that plant pathogenic oomycetes have expanded gene families that are possibly linked to their pathogenic lifestyle. We analyzed the protein domain organization of 67 eukaryotic species including four oomycete and five fungal plant pathogens. We detected 246 expanded domains in fungal and oomycete plant pathogens. The analysis of genes differentially expressed during infection revealed a significant enrichment of genes encoding expanded domains as well as signal peptides linking a substantial part of these genes to pathogenicity. Overrepresentation and clustering of domain abundance profiles revealed domains that might have important roles in host-pathogen interactions but, as yet, have not been linked to pathogenicity. The number of distinct domain combinations (bigrams) in oomycetes was significantly higher than in fungi. We identified 773 oomycete-specific bigrams, with the majority composed of domains common to eukaryotes. The analyses enabled us to link domain content to biological processes such as host-pathogen interaction, nutrient uptake, or suppression and elicitation of plant immune responses. Taken together, this study represents a comprehensive overview of the domain repertoire of fungal and oomycete plant pathogens and points to novel features like domain expansion and species-specific bigram types that could, at least partially, explain why oomycetes are such remarkable plant pathogens.

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., Author Summary Fish are an increasingly important source of animal protein globally, with aquaculture production rising dramatically over the past decade. Saprolegnia is a fungal-like oomycete and one of the most destructive fish pathogens, causing millions of dollars in losses to the aquaculture industry annually. Saprolegnia has also been linked to a worldwide decline in wild fish and amphibian populations. Here we describe the genome sequence of the first animal pathogenic oomycete and compare the genome content with the available plant pathogenic oomycetes. We found that Saprolegnia lacks the large effector families that are hallmarks of plant pathogenic oomycetes, showing evolutionary adaptation to the host. Moreover, Saprolegnia harbors pathogenesis-related genes that were derived by lateral gene transfer from the host and other animal pathogens. The retrotransposon LINE family also appears to be acquired from animal lineages. By transcriptome analysis we show a high rate of allelic variation, which reveals rapidly evolving genes and potentially adaptive evolutionary mechanisms coupled to selective pressures exerted by the animal host. The genome and transcriptome data, as well as subsequent biochemical analyses, provided us with insight in the disease process of Saprolegnia at a molecular and cellular level, providing us with targets for sustainable control of Saprolegnia.

Background 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. Results Focussing on human protein complexes, we based our analysis on a manually curated dataset from HPRD. In total, 1,060 complexes with 6,136 proteins from 2,187 unique genes were considered. We computed interologs in 25 different species and predicted the composition of complexes. Over the analysed species, the composition of most complexes was highly flexible and only 25% of all genes were never lost. Even if one component was lost at a particular point in time, the fraction of observed second, independent losses of additional components was high (75% of all complexes affected). Still, loss of whole complexes happened rarely. This biological signal deviated significantly from random models. We exemplified this trend on the anaphase promoting complex (APC) where a core is highly conserved throughout all metazoans, but flexibility in certain components is observable. Conclusion Consecutive additions and losses of distinct units is a fundamental process in the evolution of protein complexes. These evolutionary events affecting genes coding for units in human protein complexes showed a significantly different phylogenetic pattern compared to randomly selected genes. Determination of taxon specific attachments or losses might be linked to specific cellular or morphological features. Thus, protein complexes contain not only structural and functional, but also evolutionary cores.