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Fungal pathogens deploy a set of molecules (proteins, specialized metabolites, and sRNAs), so-called effectors, to aid the infection process. In comparison to other plant pathogens, smut fungi have small genomes and secretomes of 20 Mb and around 500 proteins, respectively. Previous comparative genomic studies have shown that many secreted effector proteins without known domains, i.e., novel, are conserved only in the Ustilaginaceae family. By analyzing the secretomes of 11 species within Ustilaginaceae, we identified 53 core homologous groups commonly present in this lineage. By collecting existing mutants and generating additional ones, we gathered 44 Ustilago maydis strains lacking single core effectors as well as 9 strains containing multiple deletions of core effector gene families. Pathogenicity assays revealed that 20 of these 53 mutant strains were affected in virulence. Among the 33 mutants that had no obvious phenotypic changes, 13 carried additional, sequence-divergent, structurally similar paralogs. We report a virulence contribution of seven previously uncharacterized single core effectors and of one effector family. Our results help to prioritize effectors for understanding U. maydis virulence and provide genetic resources for further characterization. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Background: Fungi are important sources for bioactive compounds that find their applications in many important sectors like in the pharma-, food- or agricultural industries. In an environmental monitoring project for fungi involved in soil nitrogen cycling we also isolated Cephalotrichum gorgonifer (strain NG_p51). In the course of strain characterisation work we found that this strain is able to naturally produce high amounts of rasfonin, a polyketide inducing autophagy, apoptosis, necroptosis in human cell lines and showing anti-tumor activity in KRAS-dependent cancer cells., Results: In order to elucidate the biosynthetic pathway of rasfonin, the strain was genome sequenced, annotated, submitted to transcriptome analysis and genetic transformation was established. Biosynthetic gene cluster (BGC) prediction revealed the existence of 22 BGCs of which the majority was not expressed under our experimental conditions. In silico prediction revealed two BGCs with a suite of enzymes possibly involved in rasfonin biosynthesis. Experimental verification by gene-knock out of the key enzyme genes showed that one of the predicted BGCs is indeed responsible for rasfonin biosynthesis., Conclusions: This study identified a biosynthetic gene cluster containing a key-gene responsible for rasfonin production. Additionally, molecular tools were established for the non-model fungus Cephalotrichum gorgonifer which allows strain engineering and heterologous expression of the BGC for high rasfonin producing strains and the biosynthesis of rasfonin derivates for diverse applications., (© 2023. The Author(s).)

Outcome research that supports guideline recommendations for primary and secondary preventions largely depends on the data obtained from clinical trials or selected hospital populations. The exponentially growing amount of real-world medical data could enable fundamental improvements in cardiovascular disease (CVD) prediction, prevention, and care. In this review we summarize how data from health insurance claims (HIC) may improve our understanding of current health provision and identify challenges of patient care by implementing the perspective of patients (providing data and contributing to society), physicians (identifying at-risk patients, optimizing diagnosis and therapy), health insurers (preventive education and economic aspects), and policy makers (data-driven legislation). HIC data has the potential to inform relevant aspects of the healthcare systems. Although HIC data inherit limitations, large sample sizes and long-term follow-up provides enormous predictive power. Herein, we highlight the benefits and limitations of HIC data and provide examples from the cardiovascular field, i.e. how HIC data is supporting healthcare, focusing on the demographical and epidemiological differences, pharmacotherapy, healthcare utilization, cost-effectiveness and outcomes of different treatments. As an outlook we discuss the potential of using HIC-based big data and modern artificial intelligence (AI) algorithms to guide patient education and care, which could lead to the development of a learning healthcare system and support a medically relevant legislation in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Krefting, Sen, David-Rus, Güldener, Hawe, Cassese, von Scheidt and Schunkert.)

Objective: Machine learning (ML) approaches have the potential to uncover regular patterns in multi-layered data. Here we applied self-organizing maps (SOMs) to detect such patterns with the aim to better predict in-stent restenosis (ISR) at surveillance angiography 6 to 8 months after percutaneous coronary intervention with stenting., Methods: In prospectively collected data from 10,004 patients receiving percutaneous coronary intervention (PCI) for 15,004 lesions, we applied SOMs to predict ISR angiographically 6-8 months after index procedure. SOM findings were compared with results of conventional uni- and multivariate analyses. The predictive value of both approaches was assessed after random splitting of patients into training and test sets (50:50)., Results: Conventional multivariate analyses revealed 10, mostly known, predictors for restenosis after coronary stenting: balloon-to-vessel ratio, complex lesion morphology, diabetes mellitus, left main stenting, stent type (bare metal vs. first vs. second generation drug eluting stent), stent length, stenosis severity, vessel size reduction, and prior bypass surgery. The SOM approach identified all these and nine further predictors, including chronic vessel occlusion, lesion length, and prior PCI. Moreover, the SOM-based model performed well in predicting ISR (AUC under ROC: 0.728); however, there was no meaningful advantage in predicting ISR at surveillance angiography in comparison with the conventional multivariable model (0.726, p = 0.3)., Conclusions: The agnostic SOM-based approach identified-without clinical knowledge-even more contributors to restenosis risk. In fact, SOMs applied to a large prospectively sampled cohort identified several novel predictors of restenosis after PCI. However, as compared with established covariates, ML technologies did not improve identification of patients at high risk for restenosis after PCI in a clinically relevant fashion.

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR-Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD., (© 2022. The Author(s).)

The majority of risk loci identified by genome-wide association studies (GWAS) are in non-coding regions, hampering their functional interpretation. Instead, transcriptome-wide association studies (TWAS) identify gene-trait associations, which can be used to prioritize candidate genes in disease-relevant tissue(s). Here, we aimed to systematically identify susceptibility genes for coronary artery disease (CAD) by TWAS. We trained prediction models of nine CAD-relevant tissues using EpiXcan based on two genetics-of-gene-expression panels, the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) and the Genotype-Tissue Expression (GTEx). Based on these prediction models, we imputed gene expression of respective tissues from individual-level genotype data on 37,997 CAD cases and 42,854 controls for the subsequent gene-trait association analysis. Transcriptome-wide significant association (i.e. P < 3.85e-6) was observed for 114 genes. Of these, 96 resided within previously identified GWAS risk loci and 18 were novel. Stepwise analyses were performed to study their plausibility, biological function, and pathogenicity in CAD, including analyses for colocalization, damaging mutations, pathway enrichment, phenome-wide associations with human data and expression-traits correlations using mouse data. Finally, CRISPR/Cas9-based gene knockdown of two newly identified TWAS genes, RGS19 and KPTN, in a human hepatocyte cell line resulted in reduced secretion of APOB100 and lipids in the cell culture medium. Our CAD TWAS work (i) prioritized candidate causal genes at known GWAS loci, (ii) identified 18 novel genes to be associated with CAD, and iii) suggested potential tissues and pathways of action for these TWAS CAD genes., (© 2022. The Author(s).)

Background: Epidemiological studies have repeatedly observed a markedly higher risk for coronary artery disease (CAD) in Scotland as compared to England. Up to now, it is unclear whether environmental or genetic factors might explain this phenomenon., Methods: Using UK Biobank (UKB) data, we assessed CAD risk, based on the Framingham risk score (FRS) and common genetic variants, to explore the respective contribution to CAD prevalence in Scotland (n = 31,963) and England (n = 317,889). We calculated FRS based on sex, age, body mass index (BMI), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), systolic blood pressure (SBP), antihypertensive medication, smoking status, and diabetes. We determined the allele frequency of published genome-wide significant risk CAD alleles and a weighted genetic risk score (wGRS) for quantifying genetic CAD risk., Results: Prevalence of CAD was 16% higher in Scotland as compared to England (8.98% vs. 7.68%, P < 0.001). However, the FRS only predicted a marginally higher CAD risk (less than 1%) in Scotland (12.5 ± 10.5 vs.12.6 ± 10.6, P = 0.03). Likewise, the overall number of genome-wide significant variants affecting CAD risk (157.6 ± 7.7 and 157.5 ± 7.7; P = 0.12) and a wGRS for CAD (2.49 ± 0.25 in both populations, P = 0.14) were remarkably similar in the English and Scottish population. Interestingly, we observed substantial differences in the allele frequencies of individual risk variants. Of the previously described 163 genome-wide significant variants studied here, 35 variants had higher frequencies in Scotland, whereas 37 had higher frequencies in England (P < 0.001 each)., Conclusions: Neither the traditional risk factors included in the FRS nor a genetic risk score (GRS) based on established common risk alleles explained the higher CAD prevalence in Scotland. However, we observed marked differences in the distribution of individual risk alleles, which emphasizes that even geographically and ethnically closely related populations may display relevant differences in the genetic architecture of a common disease., (© 2021. The Author(s).)

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Non-coding RNAs have already been linked to CVD development and progression. While microRNAs (miRs) have been well studied in blood samples, there is little data on tissue-specific miRs in cardiovascular relevant tissues and their relation to cardiovascular risk factors. Tissue-specific miRs derived from Arteria mammaria interna (IMA) from 192 coronary artery disease (CAD) patients undergoing coronary artery bypass grafting (CABG) were analyzed. The aims of the study were 1) to establish a reference atlas which can be utilized for identification of novel diagnostic biomarkers and potential therapeutic targets, and 2) to relate these miRs to cardiovascular risk factors. Overall, 393 individual miRs showed sufficient expression levels and passed quality control for further analysis. We identified 17 miRs-miR-10b-3p, miR-10-5p, miR-17-3p, miR-21-5p, miR-151a-5p, miR-181a-5p, miR-185-5p, miR-194-5p, miR-199a-3p, miR-199b-3p, miR-212-3p, miR-363-3p, miR-548d-5p, miR-744-5p, miR-3117-3p, miR-5683 and miR-5701-significantly correlated with cardiovascular risk factors (correlation coefficient >0.2 in both directions, p -value ( p < 0.006, false discovery rate (FDR) <0.05). Of particular interest, miR-5701 was positively correlated with hypertension, hypercholesterolemia, and diabetes. In addition, we found that miR-629-5p and miR-98-5p were significantly correlated with acute myocardial infarction. We provide a first atlas of miR profiles in IMA samples from CAD patients. In perspective, these miRs might play an important role in improved risk assessment, mechanistic disease understanding and local therapy of CAD.

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 ( FUP2 , FPRN&#95;05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN&#95;05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase ( FUP4 , FPRN&#95;05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase ( FUP5 , FPRN&#95;05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

Background: Saccharomycodes ludwigii belongs to the poorly characterized Saccharomycodeacea family and is known by its ability to spoil wines, a trait mostly attributable to its high tolerance to sulfur dioxide (SO 2 ). To improve knowledge about Saccharomycodeacea our group determined whole-genome sequences of Hanseniaspora guilliermondii (UTAD222) and S. ludwigii (UTAD17), two members of this family. While in the case of H. guilliermondii the genomic information elucidated crucial aspects concerning the physiology of this species in the context of wine fermentation, the draft sequence obtained for S. ludwigii was distributed by more than 1000 contigs complicating extraction of biologically relevant information. In this work we describe the results obtained upon resequencing of S. ludwigii UTAD17 genome using PacBio as well as the insights gathered from the exploration of the annotation performed over the assembled genome., Results: Resequencing of S. ludwigii UTAD17 genome with PacBio resulted in 20 contigs totaling 13 Mb of assembled DNA and corresponding to 95% of the DNA harbored by this strain. Annotation of the assembled UTAD17 genome predicts 4644 protein-encoding genes. Comparative analysis of the predicted S. ludwigii ORFeome with those encoded by other Saccharomycodeacea led to the identification of 213 proteins only found in this species. Among these were six enzymes required for catabolism of N-acetylglucosamine, four cell wall β-mannosyltransferases, several flocculins and three acetoin reductases. Different from its sister Hanseniaspora species, neoglucogenesis, glyoxylate cycle and thiamine biosynthetic pathways are functional in S. ludwigii. Four efflux pumps similar to the Ssu1 sulfite exporter, as well as robust orthologues for 65% of the S. cerevisiae SO 2 -tolerance genes, were identified in S. ludwigii genome., Conclusions: This work provides the first genome-wide picture of a S. ludwigii strain representing a step forward for a better understanding of the physiology and genetics of this species and of the Saccharomycodeacea family. The release of this genomic sequence and of the information extracted from it can contribute to guide the design of better wine preservation strategies to counteract spoilage prompted by S. ludwigii. It will also accelerate the exploration of this species as a cell factory, specially in production of fermented beverages where the use of Non-Saccharomyces species (including spoilage species) is booming.

Background: Epidemiological studies have shown inverse association between intelligence and coronary artery disease (CAD) risk, but the underlying mechanisms remain unclear., Methods: Based on 242 SNPs independently associated with intelligence, we calculated the genetic intelligence score (gIQ) for participants from 10 CAD case-control studies (n = 34,083) and UK Biobank (n = 427,306). From UK Biobank, we extracted phenotypes including body mass index (BMI), type 2 diabetes (T2D), smoking, hypertension, HDL cholesterol, LDL cholesterol, measured intelligence score, and education attainment. To estimate the effects of gIQ on CAD and its related risk factors, regression analyses was applied. Next, we studied the mediatory roles of measured intelligence and educational attainment. Lastly, Mendelian randomization was performed to validate the findings., Results: In CAD case-control studies, one standard deviation (SD) increase of gIQ was related to a 5% decrease of CAD risk (odds ratio [OR] of 0.95; 95% confidence interval [CI] 0.93 to 0.98; P = 4.93e-5), which was validated in UK Biobank (OR = 0.97; 95% CI 0.96 to 0.99; P = 6.4e-4). In UK Biobank, we also found significant inverse correlations between gIQ and risk factors of CAD including smoking, BMI, T2D, hypertension, and a positive correlation with HDL cholesterol. The association signals between gIQ and CAD as well as its risk factors got largely attenuated after the adjustment of measured intelligence and educational attainment. The causal role of intelligence in mediating CAD risk was confirmed by Mendelian randomization analyses., Conclusion: Genetic components of intelligence affect measured intelligence and educational attainment, which subsequently affect the prevalence of CAD via a series of unfavorable risk factor profiles.

A missense variant of the sushi, von Willebrand factor type A, EGF and pentraxin domain containing protein 1 (SVEP1) is genome-wide significantly associated with coronary artery disease. The mechanisms how SVEP1 impacts atherosclerosis are not known. We found endothelial cells (EC) and vascular smooth muscle cells to represent the major cellular source of SVEP1 in plaques. Plaques were larger in atherosclerosis-prone Svep1 haploinsufficient (ApoE -/- Svep1 +/- ) compared to Svep1 wild-type mice (ApoE -/- Svep1 +/+ ) and ApoE -/- Svep1 +/- mice displayed elevated plaque neutrophil, Ly6C high monocyte, and macrophage numbers. We assessed how leukocytes accumulated more inside plaques in ApoE -/- Svep1 +/- mice and found enhanced leukocyte recruitment from blood into plaques. In vitro, we examined how SVEP1 deficiency promotes leukocyte recruitment and found elevated expression of the leukocyte attractant chemokine (C-X-C motif) ligand 1 (CXCL1) in EC after incubation with missense compared to wild-type SVEP1. Increasing wild-type SVEP1 levels silenced endothelial CXCL1 release. In line, plasma Cxcl1 levels were elevated in ApoE -/- Svep1 +/- mice. Our studies reveal an atheroprotective role of SVEP1. Deficiency of wild-type Svep1 increased endothelial CXCL1 expression leading to enhanced recruitment of proinflammatory leukocytes from blood to plaque. Consequently, elevated vascular inflammation resulted in enhanced plaque progression in Svep1 deficiency.

Fusarium graminearum is one of the most destructive plant pathogens worldwide, causing fusarium head blight (FHB) on cereals. F. graminearum colonizes wheat plant surfaces with specialized unbranched hyphae called runner hyphae (RH), which develop multicelled complex appressoria called infection cushions (IC). IC generate multiple penetration sites, allowing the fungus to enter the plant cuticle. Complex infection structures are typical for several economically important plant pathogens, yet with unknown molecular basis. In this study, RH and IC formed on the surface of wheat paleae were isolated by laser capture microdissection. RNA-Seq-based transcriptomic analyses were performed on RH and IC and compared to mycelium grown in complete medium (MY). Both RH and IC displayed a high number of infection up-regulated genes (982), encoding, among others, carbohydrate-active enzymes (CAZymes: 140), putative effectors (PE: 88), or secondary metabolism gene clusters (SMC: 12 of 67 clusters). RH specifically up-regulated one SMC corresponding to aurofusarin biosynthesis, a broad activity antibiotic. IC specifically up-regulated 248 genes encoding mostly putative virulence factors such as 7 SMC, including the mycotoxin deoxynivalenol and the newly identified fusaoctaxin A, 33 PE, and 42 CAZymes. Furthermore, we studied selected candidate virulence factors using cellular biology and reverse genetics. Hence, our results demonstrate that IC accumulate an arsenal of proven and putative virulence factors to facilitate the invasion of epidermal cells., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Pathogenic fungi can have devastating effects on agriculture and health. One potential challenge in dealing with pathogens is the possibility of a host jump (i.e., when a pathogen infects a new host species). This can lead to the emergence of new diseases or complicate the management of existing threats. We studied host specificity by using a hybrid fungus formed by mating two closely related fungi: Ustilago bromivora , which normally infects Brachypodium spp., and U. hordei , which normally infects barley. Although U. hordei was unable to infect Brachypodium spp., the hybrid could. These hybrids also displayed the same mating-type bias that had been observed in U. bromivora and provide evidence of a dominant spore-killer-like system on the sex chromosome of U. bromivora . By analyzing the genomic composition of 109 hybrid strains, backcrossed with U. hordei over four generations, we identified three regions associated with infection on Brachypodium spp. and 75 potential virulence candidates. The most strongly associated region was located on chromosome 8, where seven genes encoding predicted secreted proteins were identified. The fact that we identified several regions relevant for pathogenicity on Brachypodium spp. but that none were essential suggests that host specificity, in the case of U. bromivora , is a multifactorial trait which can be achieved through different subsets of virulence factors.

Fusarium graminearum is a plant pathogenic fungus which is able to infect wheat and other economically important cereal crop species. The role of ethylene in the interaction with host plants is unclear and controversial. We have analyzed the inventory of genes with a putative function in ethylene production or degradation of the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC). F. graminearum , in contrast to other species, does not contain a candidate gene encoding ethylene-forming enzyme. Three genes with similarity to ACC synthases exist; heterologous expression of these did not reveal enzymatic activity. The F. graminearum genome contains in addition two ACC deaminase candidate genes. We have expressed both genes in E. coli and characterized the enzymatic properties of the affinity-purified products. One of the proteins had indeed ACC deaminase activity, with kinetic properties similar to ethylene-stress reducing enzymes of plant growth promoting bacteria. The other candidate was inactive with ACC but turned out to be a d-cysteine desulfhydrase. Since it had been reported that ethylene insensitivity in transgenic wheat increased Fusarium resistance and reduced the content of the mycotoxin deoxynivalenol (DON) in infected wheat, we generated single and double knockout mutants of both genes in the F. graminearum strain PH-1. No statistically significant effect of the gene disruptions on fungal spread or mycotoxin content was detected, indicating that the ability of the fungus to manipulate the production of the gaseous plant hormones ethylene and H 2 S is dispensable for full virulence.

Hanseanispora species, including H. guilliermondii, are long known to be abundant in wine grape-musts and to play a critical role in vinification by modulating, among other aspects, the wine sensory profile. Despite this, the genetics and physiology of Hanseniaspora species remains poorly understood. The first genomic sequence of a H. guilliermondii strain (UTAD222) and the discussion of its potential significance are presented in this work. Metabolic reconstruction revealed that H. guilliermondii is not equipped with a functional gluconeogenesis or glyoxylate cycle, nor does it harbours key enzymes for glycerol or galactose catabolism or for biosynthesis of biotin and thiamine. Also, no fructose-specific transporter could also be predicted from the analysis of H. guilliermondii genome leaving open the mechanisms underlying the fructophilic character of this yeast. Comparative analysis involving H. guilliermondii, H. uvarum, H. opuntiae and S. cerevisiae revealed 14 H. guilliermondii-specific genes (including five viral proteins and one β-glucosidase). Furthermore, 870 proteins were only found within the Hanseniaspora proteomes including several β-glucosidases and decarboxylases required for catabolism of biogenic amines. The release of H. guilliermondii genomic sequence and the comparative genomics/proteomics analyses performed, is expected to accelerate research focused on Hanseniaspora species and to broaden their application in the wine industry and in other bio-industries in which they could be explored as cell factories., (© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

It has long been known that hormones affect the interaction of a phytopathogen with its host plant. The pathogen can cause changes in plant hormone homeostasis directly by affecting biosynthesis or metabolism in the plant or by synthesizing and secreting the hormone itself. We previously demonstrated that pathogenic fungi of the Fusarium species complex are able to produce three major types of hormones: auxins, cytokinins, and gibberellins. In this work, we explore changes in the levels of these hormones in maize and mango plant tissues infected with Fusarium . The ability to produce individual phytohormones varies significantly across Fusarium species and such differences likely impact host specificity inducing the unique responses noted in planta during infection. For example, the production of gibberellins by F. fujikuroi leads to elongated rice stalks and the suppression of gibberellin biosynthesis in plant tissue. Although all Fusarium species are able to synthesize auxin, sometimes by multiple pathways, the ratio of its free form and conjugates in infected tissue is affected more than the total amount produced. The recently characterized unique pathway for cytokinin de novo synthesis in Fusarium appears silenced or non-functional in all studied species during plant infection. Despite this, a large increase in cytokinin levels was detected in F. mangiferae infected plants, caused likely by the up-regulation of plant genes responsible for their biosynthesis. Thus, the accumulation of active cytokinins may contribute to mango malformation of the reproductive organs upon infection of mango trees. Together, our findings provide insight into the complex role fungal and plant derived hormones play in the fungal-plant interactions.

Ramularia leaf spot has recently emerged as a major threat to barley production world-wide, causing 25% yield loss in many barley growing regions. Here, we provide a new reference genome of the causal agent, the Dothideomycete Ramularia collo-cygni. The assembly of 32 Mb consists of 78 scaffolds. We used RNA-seq to identify 11,622 genes of which 1,303 and 282 are coding for predicted secreted proteins and putative effectors respectively.The pathogen separated from its nearest sequenced relative, Zymoseptoria tritici ∼27 Ma. We calculated the divergence of the two species on protein level and see remarkably high synonymous and nonsynonymous divergence. Unlike in many other plant pathogens, the comparisons of transposable elements and gene distributions, show a very homogeneous genome for R. collo-cygni. We see no evidence for higher selective pressure on putative effectors or other secreted proteins and repetitive sequences are spread evenly across the scaffolds. These findings could be associated to the predominantly endophytic life-style of the pathogen. We hypothesize that R. collo-cygni only recently became pathogenic and that therefore its genome does not yet show the typical pathogen characteristics. Because of its high scaffold length and improved CDS annotations, our new reference sequence provides a valuable resource for the community for future comparative genomics and population genetics studies.

This work describes, for the first time, the genome sequence of a Saccharomycodes ludwigii strain. Although usually seen as a wine spoilage yeast, S. ludwigii has been of interest for the production of fermented beverages because it harbors several interesting properties, including the production of beneficial aroma compounds.

Here we present the identification and characterization of the H3K4-specific histone methyltransferase Set1 and its counterpart, the Jumonji C demethylase Kdm5, in the rice pathogen Fusarium fujikuroi. While Set1 is responsible for all detectable H3K4me2/me3 in this fungus, Kdm5 antagonizes the H3K4me3 mark. Notably, deletion of both SET1 and KDM5 mainly resulted in the upregulation of genome-wide transcription, also affecting a large set of secondary metabolite (SM) key genes. Although H3K4 methylation is a hallmark of actively transcribed euchromatin, several SM gene clusters located in subtelomeric regions were affected by Set1 and Kdm5. While the regulation of many of them is likely indirect, H3K4me2 levels at gibberellic acid (GA) genes correlated with GA biosynthesis in the wild type, Δkdm5 and OE::KDM5 under inducing conditions. Whereas Δset1 showed an abolished GA 3 production in axenic culture, phytohormone biosynthesis was induced in planta, so that residual amounts of GA 3 were detected during rice infection. Accordingly, Δset1 exhibited a strongly attenuated, though not abolished, virulence on rice. Apart from regulating secondary metabolism, Set1 and Kdm5 function as activator and repressor of conidiation respectively. They antagonistically regulate H3K4me3 levels and expression of the major conidiation-specific transcription factor gene ABA1 in F. fujikuroi., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

There is a need for flexible and affordable plant phenotyping solutions for basic research and plant breeding. We demonstrate our open source plant imaging and processing solution ('PhenoBox'/'PhenoPipe') and provide construction plans, source code and documentation to rebuild the system. Use of the PhenoBox is exemplified by studying infection of the model grass Brachypodium distachyon by the head smut fungus Ustilago bromivora, comparing phenotypic responses of maize to infection with a solopathogenic Ustilago maydis (corn smut) strain and effector deletion strains, and studying salt stress response in Nicotiana benthamiana. In U. bromivora-infected grass, phenotypic differences between infected and uninfected plants were detectable weeks before qualitative head smut symptoms. Based on this, we could predict the infection outcome for individual plants with high accuracy. Using a PhenoPipe module for calculation of multi-dimensional distances from phenotyping data, we observe a time after infection-dependent impact of U. maydis effector deletion strains on phenotypic response in maize. The PhenoBox/PhenoPipe system is able to detect established salt stress responses in N. benthamiana. We have developed an affordable, automated, open source imaging and data processing solution that can be adapted to various phenotyping applications in plant biology and beyond., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

In the version of this Article originally published, it was incorrectly stated that "16,687 protein-coding genes were inferred for the most recent common ancestor (MRCA) of Armillaria"; the value was incorrect and it should have read "15,787". This has now been corrected.

The frequent emergence of azole resistance among Candida glabrata strains contributes to increase the incidence of infections caused by this species. Whole-genome sequencing of a fluconazole and voriconazole-resistant clinical isolate (FFUL887) and subsequent comparison with the genome of the susceptible strain CBS138 revealed prominent differences in several genes documented to promote azole resistance in C. glabrata. Among these was the transcriptional regulator CgPdr1. The CgPdr1 FFUL887 allele included a K274Q modification not documented in other azole-resistant strains. Transcriptomic profiling evidenced the upregulation of 92 documented targets of CgPdr1 in the FFUL887 strain, supporting the idea that the K274Q substitution originates a CgPdr1 gain-of-function mutant. The expression of CgPDR1K274Q in the FFUL887 background sensitised the cells against high concentrations of organic acids at a low pH (4.5), but had no detectable effect in tolerance towards other environmental stressors. Comparison of the genome of FFUL887 and CBS138 also revealed prominent differences in the sequence of adhesin-encoding genes, while comparison of the transcriptome of the two strains showed a significant remodelling of the expression of genes involved in metabolism of carbohydrates, nitrogen and sulphur in the FFUL887 strain; these responses likely reflecting adaptive responses evolved by the clinical strain during colonisation of the host., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), a family of plant hormones. Recent genome sequencing revealed the genetic capacity for the biosynthesis of 46 additional secondary metabolites besides the industrially produced GAs. Among them are the pigments bikaverin and fusarubins, as well as mycotoxins, such as fumonisins, fusarin C, beauvericin, and fusaric acid. However, half of the potential secondary metabolite gene clusters are silent. In recent years, it has been shown that the fungal specific velvet complex is involved in global regulation of secondary metabolism in several filamentous fungi. We have previously shown that deletion of the three components of the F. fujikuroi velvet complex, vel1, vel2, and lae1, almost totally abolished biosynthesis of GAs, fumonisins and fusarin C. Here, we present a deeper insight into the genome-wide regulatory impact of Lae1 on secondary metabolism. Over-expression of lae1 resulted in de-repression of GA biosynthetic genes under otherwise repressing high nitrogen conditions demonstrating that the nitrogen repression is overcome. In addition, over-expression of one of five tested histone acetyltransferase genes, HAT1, was capable of returning GA gene expression and GA production to the GA-deficient Δlae1 mutant. Deletion and over-expression of HAT1 in the wild type resulted in downregulation and upregulation of GA gene expression, respectively, indicating that HAT1 together with Lae1 plays an essential role in the regulation of GA biosynthesis.

In this work, we present a comprehensive analysis of the H3K36 histone methyltransferases Set2 and Ash1 in the filamentous ascomycete Fusarium fujikuroi In Saccharomyces cerevisiae , one single methyltransferase, Set2, confers all H3K36 methylation, while there are two members of the Set2 family in filamentous fungi, and even more H3K36 methyltransferases in higher eukaryotes. Whereas the yeast Set2 homolog has been analyzed in fungi previously, the second member of the Set2 family, designated Ash1, has not been described for any filamentous fungus. Western blot and ChIP-Seq analyses confirmed that F. fujikuroi Set2 and Ash1 are H3K36-specific histone methyltransferases that deposit H3K36me3 at specific loci: Set2 is most likely responsible for H3K36 methylation of euchromatic regions of the genome, while Ash1 methylates H3K36 at the subtelomeric regions (facultative heterochromatin) of all chromosomes, including the accessory chromosome XII. Our data indicate that H3K36me3 cannot be considered a hallmark of euchromatin in F. fujikuroi , and likely also other filamentous fungi, making them different to what is known about nuclear characteristics in yeast and higher eukaryotes. We suggest that the H3K36 methylation mark exerts specific functions when deposited at euchromatic or subtelomeric regions by Set2 or Ash1, respectively. We found an enhanced level of H3K27me3, an increased instability of subtelomeric regions and losses of the accessory chromosome XII over time in Δ ash1 mutants, indicating an involvement of Ash1 in DNA repair processes. Further phenotypic analyses revealed a role of H3K36 methylation in vegetative growth, sporulation, secondary metabolite biosynthesis, and virulence in F. fujikuroi ., (Copyright © 2018 by the Genetics Society of America.)

Armillaria species are both devastating forest pathogens and some of the largest terrestrial organisms on Earth. They forage for hosts and achieve immense colony sizes via rhizomorphs, root-like multicellular structures of clonal dispersal. Here, we sequenced and analysed the genomes of four Armillaria species and performed RNA sequencing and quantitative proteomic analysis on the invasive and reproductive developmental stages of A. ostoyae. Comparison with 22 related fungi revealed a significant genome expansion in Armillaria, affecting several pathogenicity-related genes, lignocellulose-degrading enzymes and lineage-specific genes expressed during rhizomorph development. Rhizomorphs express an evolutionarily young transcriptome that shares features with the transcriptomes of both fruiting bodies and vegetative mycelia. Several genes show concomitant upregulation in rhizomorphs and fruiting bodies and share cis-regulatory signatures in their promoters, providing genetic and regulatory insights into complex multicellularity in fungi. Our results suggest that the evolution of the unique dispersal and pathogenicity mechanisms of Armillaria might have drawn upon ancestral genetic toolkits for wood-decay, morphogenesis and complex multicellularity.

Filamentous fungi exhibit a broad spectrum of heritable growth patterns and morphological variations reflecting the adaptation of the different species to distinct ecological niches. But also within species, isolates show considerable variation in growth rates and other morphological characteristics. The genetic basis of this intraspecific variation in mycelial growth and morphology is currently poorly understood. By chance, a growth mutant in the root rot pathogen Armillaria ostoyae was discovered. The mutant phenotype was characterized by extremely compact and slow growth, as well as shorter aerial hyphae and hyphal compartments in comparison to the wildtype phenotype. Genetic analysis revealed that the abnormal phenotype is caused by a recessive mutation, which segregates asa single locus in sexual crosses. In order to identify the genetic basis of the mutant phenotype, we performed a quantitative trait locus (QTL) analysis. A mapping population of 198 haploid progeny was genotyped at 11,700 genome-wide single nucleotide polymorphisms (SNPs) making use of double digest restriction site associated DNA sequencing (ddRADseq). In accordance with the genetic analysis, a single significant QTL was identified for the abnormal growth phenotype. The QTL confidence interval spans a narrow, gene dense region of 87kb in the A. ostoyae genome which contains 37 genes. Overall, our study reports the first high-density genetic map for an Armillaria species and shows its successful application in forward genetics by resolving the genetic basis of a mutant phenotype with a severe defect in hyphal growth., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Fusarium fujikuroi causes bakanae ("foolish seedling") disease of rice which is characterized by hyper-elongation of seedlings resulting from production of gibberellic acids (GAs) by the fungus. This plant pathogen is also known for production of harmful mycotoxins, such as fusarins, fusaric acid, apicidin F and beauvericin. Recently, we generated the first de novo genome sequence of F. fujikuroi strain IMI 58289 combined with extensive transcriptional, epigenetic, proteomic and chemical product analyses. GA production was shown to provide a selective advantage during infection of the preferred host plant rice. Here, we provide genome sequences of eight additional F. fujikuroi isolates from distant geographic regions. The isolates differ in the size of chromosomes, most likely due to variability of subtelomeric regions, the type of asexual spores (microconidia and/or macroconidia), and the number and expression of secondary metabolite gene clusters. Whilst most of the isolates caused the typical bakanae symptoms, one isolate, B14, caused stunting and early withering of infected seedlings. In contrast to the other isolates, B14 produced no GAs but high amounts of fumonisins during infection on rice. Furthermore, it differed from the other isolates by the presence of three additional polyketide synthase (PKS) genes (PKS40, PKS43, PKS51) and the absence of the F. fujikuroi-specific apicidin F (NRPS31) gene cluster. Analysis of additional field isolates confirmed the strong correlation between the pathotype (bakanae or stunting/withering), and the ability to produce either GAs or fumonisins. Deletion of the fumonisin and fusaric acid-specific PKS genes in B14 reduced the stunting/withering symptoms, whereas deletion of the PKS51 gene resulted in elevated symptom development. Phylogenetic analyses revealed two subclades of F. fujikuroi strains according to their pathotype and secondary metabolite profiles.

Zygosaccharomyces bailii is one of the most problematic spoilage yeast species found in the food and beverage industry particularly in acidic products, due to its exceptional resistance to weak acid stress. This article describes the annotation of the genome sequence of Z. bailii IST302, a strain recently proven to be amenable to genetic manipulations and physiological studies. The work was based on the annotated genomes of strain ISA1307, an interspecies hybrid between Z. bailii and a closely related species, and the Z. bailii reference strain CLIB 213T. The resulting genome sequence of Z. bailii IST302 is distributed through 105 scaffolds, comprising a total of 5142 genes and a size of 10.8 Mb. Contrasting with CLIB 213T, strain IST302 does not form cell aggregates, allowing its manipulation in the laboratory for genetic and physiological studies. Comparative cell cycle analysis with the haploid and diploid Saccharomyces cerevisiae strains BY4741 and BY4743, respectively, suggests that Z. bailii IST302 is haploid. This is an additional trait that makes this strain attractive for the functional analysis of non-essential genes envisaging the elucidation of mechanisms underlying its high tolerance to weak acid food preservatives, or the investigation and exploitation of the potential of this resilient yeast species as cell factory., (© FEMS 2017.)

The biosynthesis of multiple secondary metabolites in the phytopathogenic ascomycete Fusarium fujikuroi is strongly affected by nitrogen availability. Here, we present the first genome-wide transcriptome and proteome analysis that compared the wild type and deletion mutants of the two major nitrogen regulators AreA and AreB. We show that AreB acts not simply as an antagonist of AreA counteracting the expression of AreA target genes as suggested based on the yeast model. Both GATA transcription factors affect a large and diverse set of common as well as specific target genes and proteins, acting as activators and repressors. We demonstrate that AreA and AreB are not only involved in fungal nitrogen metabolism, but also in the control of several complex cellular processes like carbon metabolism, transport and secondary metabolism. We show that both GATA transcription factors can be considered as master regulators of secondary metabolism as they affect the expression of more than half of the 47 putative secondary metabolite clusters identified in the genome of F. fujikuroi. While AreA acts as a positive regulator of many clusters under nitrogen-limiting conditions, AreB is able to activate and repress gene clusters (e.g. bikaverin) under nitrogen limitation and sufficiency. In addition, ChIP analyses revealed that loss of AreA or AreB causes histone modifications at some of the regulated gene clusters.

In this work, we disclose the genome sequence and a corresponding manually curated annotation of the non-Saccharomyces yeast Hanseniaspora guilliermondii UTAD222, a strain shown to have interesting oenological traits for the production of wines with improved aromatic properties., (Copyright © 2017 Seixas et al.)

Species of the Fusarium fujikuroi species complex (FFC) cause a wide spectrum of often devastating diseases on diverse agricultural crops, including coffee, fig, mango, maize, rice, and sugarcane. Although species within the FFC are difficult to distinguish by morphology, and their genes often share 90% sequence similarity, they can differ in host plant specificity and life style. FFC species can also produce structurally diverse secondary metabolites (SMs), including the mycotoxins fumonisins, fusarins, fusaric acid, and beauvericin, and the phytohormones gibberellins, auxins, and cytokinins. The spectrum of SMs produced can differ among closely related species, suggesting that SMs might be determinants of host specificity. To date, genomes of only a limited number of FFC species have been sequenced. Here, we provide draft genome sequences of three more members of the FFC: a single isolate of F. mangiferae, the cause of mango malformation, and two isolates of F. proliferatum, one a pathogen of maize and the other an orchid endophyte. We compared these genomes to publicly available genome sequences of three other FFC species. The comparisons revealed species-specific and isolate-specific differences in the composition and expression (in vitro and in planta) of genes involved in SM production including those for phytohormome biosynthesis. Such differences have the potential to impact host specificity and, as in the case of F. proliferatum, the pathogenic versus endophytic life style., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Whereas an increasing number of pathogenic and mutualistic ascomycetous species were sequenced in the past decade, species showing a seemingly neutral association such as root endophytes received less attention. In the present study, the genome of Phialocephala subalpina, the most frequent species of the Phialocephala fortinii s.l. - Acephala applanata species complex, was sequenced for insight in the genome structure and gene inventory of these wide-spread root endophytes., Results: The genome of P. subalpina was sequenced using Roche/454 GS FLX technology and a whole genome shotgun strategy. The assembly resulted in 205 scaffolds and a genome size of 69.7 Mb. The expanded genome size in P. subalpina was not due to the proliferation of transposable elements or other repeats, as is the case with other ascomycetous genomes. Instead, P. subalpina revealed an expanded gene inventory that includes 20,173 gene models. Comparative genome analysis of P. subalpina with 13 ascomycetes shows that P. subalpina uses a versatile gene inventory including genes specific for pathogens and saprophytes. Moreover, the gene inventory for carbohydrate active enzymes (CAZymes) was expanded including genes involved in degradation of biopolymers, such as pectin, hemicellulose, cellulose and lignin., Conclusions: The analysis of a globally distributed root endophyte allowed detailed insights in the gene inventory and genome organization of a yet largely neglected group of organisms. We showed that the ubiquitous root endophyte P. subalpina has a broad gene inventory that links pathogenic and saprophytic lifestyles.

Background: The Rhynchosporium species complex consists of hemibiotrophic fungal pathogens specialized to different sweet grass species including the cereal crops barley and rye. A sexual stage has not been described, but several lines of evidence suggest the occurrence of sexual reproduction. Therefore, a comparative genomics approach was carried out to disclose the evolutionary relationship of the species and to identify genes demonstrating the potential for a sexual cycle. Furthermore, due to the evolutionary very young age of the five species currently known, this genus appears to be well-suited to address the question at the molecular level of how pathogenic fungi adapt to their hosts., Results: The genomes of the different Rhynchosporium species were sequenced, assembled and annotated using ab initio gene predictors trained on several fungal genomes as well as on Rhynchosporium expressed sequence tags. Structures of the rDNA regions and genome-wide single nucleotide polymorphisms provided a hypothesis for intra-genus evolution. Homology screening detected core meiotic genes along with most genes crucial for sexual recombination in ascomycete fungi. In addition, a large number of cell wall-degrading enzymes that is characteristic for hemibiotrophic and necrotrophic fungi infecting monocotyledonous hosts were found. Furthermore, the Rhynchosporium genomes carry a repertoire of genes coding for polyketide synthases and non-ribosomal peptide synthetases. Several of these genes are missing from the genome of the closest sequenced relative, the poplar pathogen Marssonina brunnea, and are possibly involved in adaptation to the grass hosts. Most importantly, six species-specific genes coding for protein effectors were identified in R. commune. Their deletion yielded mutants that grew more vigorously in planta than the wild type., Conclusion: Both cryptic sexuality and secondary metabolites may have contributed to host adaptation. Most importantly, however, the growth-retarding activity of the species-specific effectors suggests that host adaptation of R. commune aims at extending the biotrophic stage at the expense of the necrotrophic stage of pathogenesis. Like other apoplastic fungi Rhynchosporium colonizes the intercellular matrix of host leaves relatively slowly without causing symptoms, reminiscent of the development of endophytic fungi. Rhynchosporium may therefore become an object for studying the mutualism-parasitism transition.

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover, our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver., Competing Interests: The authors declare that no competing interests exist.