Biology subjects

Stukenbrock, E. H.

Publications and source records attributed to Stukenbrock, E. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Conserved protein folds underpin the diversification of secreted proteins in a fungal pathogen

BACKGROUNDDuring host colonization, fungal plant pathogens secrete effector-like proteins that alter host cell physiology and target plant-associated microbes. However, rapid evolution and low sequence conservation hinder the study and characterization of these proteins. The fungus Zymoseptoria passerinii infects Hordeum spp. and includes lineages adapted to wild and domesticated barley. To date, the evolution of effector-like proteins in this species has not been addressed. RESULTSWe combined a set of protein structure-based and network analyses to unravel the secretome of Z. passerinii. We first compared AlphaFold2 and ESMFold predictions to establish the baseline for structural analyses. We identified 72 structural clusters in the secretome, revealing fold-level relationships across divergent sequences. We showed that effector-like proteins with host putative immune-interfering functions evolved from a limited group of protein folds, whereas proteins with predicted antimicrobial properties were distributed across fold groups. Physicochemical comparisons indicate that antimicrobial effectors predominantly emerged through amino acid replacements on common effector-enriched scaffolds in Z. passerinii, reconfiguring surface charge and electrostatics. We analyzed intra- and interspecific structural variation in selected effector-enriched families by comparing Z. passerinii proteins and homologs in the sister species Z. tritici. We describe high structural similarity in core folds, with local variation in loop and surface-exposed regions, consistent with fold stability still enabling functional diversification. CONCLUSIONSThe secretome of Z. passerinii is organized around common structural folds that support diverse biological roles, including host manipulation and host-associated microbial interactions. Conserved scaffolds combined with local physicochemical variation likely contribute to rapid adaptive evolution in Z. passerinii.

genomics↗

bioRxiv2025

Molecular mimicry of plant cell-surface immune receptors by fungal secreted leucine-rich repeat proteins

O_LILeucine-rich repeat (LRR) receptor-like kinases (LRR-RLKs) are important plant immunity proteins. The wheat pathogen Zymoseptoria tritici produces many virulence effectors during infection; however, most remain uncharacterised. We identified a secreted protein from Z. tritici (ZtLRR) that consists of a single LRR domain and hypothesised that it mimics host LRR-RLKs to suppress plant immunity. C_LIO_LIWe used transient expression to probe ZtLRR function related to production of reactive oxygen species (ROS) and cell death, two important immune processes. We used AlphaFold structural predictions with targeted yeast two-hybrid to demonstrate protein-protein interactions. Transgenic wheat allowed assessment of effector function in the natural host. C_LIO_LIZtLRR suppressed ROS production and cell death in N. benthamiana with high potency. Structural predictions suggested high similarity to several plant LRR-RLKs and interaction with TaBAK1 was confirmed by targeted yeast two-hybrid. Transgenic wheat expressing ZtLRR was impaired in PAMP-triggered immunity (PTI) and had increased susceptibility to infection with compatible Z. tritici. We identified structural orthologs of ZtLRR in diverse fungal lineages and demonstrated that several of these proteins have similar immune-suppressing properties as ZtLRR. C_LIO_LIOur work demonstrates that molecular mimicry of host LRR-RLKs by phytopathogen sLRR effectors is effective at disrupting host immune pathways. C_LI

molecular biology↗

bioRxiv2025

Host-specific fungal plant pathogens exhibit distinct interactions with the leaf microbiota of wild grasses

The plants microbiome is influenced by the plant species and biotic factors such as the infection by pathogens. Pathogen-microbiome interactions are relevant for the progression of the disease since both can compete within the plant host. We hypothesize that pathogens specialized to different hosts have distinct, direct, and indirect influence on the host microbiome. We focused on the host-specific leaf pathogens Zymoseptoria tritici and Zymoseptoria passerinii. By using microbiome metabarcoding and coculture interactions, we evaluated the influence of virulent (wild host-infecting pathogen) and avirulent (domesticated host-infecting pathogen) Zymoseptoria lineages on the leaf microbiome of the wild grasses Aegilops cylindrica and Hordeum murinum which are hosts to virulent lineages of Z. tritici and Z. passerinii, respectively. Our microbiome analysis showed that the fungal communities were affected by virulent lineages, while the avirulent lineages had the most negative correlations with bacteria. Both virulent and avirulent pathogens had the same spectrum of interactions when experimentally cocultured with bacteria. The intensity of pathogen-induced growth enhancement differed between Zymoseptoria lineages. We demonstrated that sugar metabolism through the fungal secretion of invertase can be a determinant of bacterial growth enhancement. Our study highlights the role of microbial interactions on host-specificity and mechanisms underlying microbial interactions by Zymoseptoria spp.

microbiology↗

bioRxiv2024

Ecological assembly dynamics of the seed-borne microbiome in cultivated and wild wheat

BackgroundPlants host diverse microbial communities that can significantly impact plant health. A portion of these communities is vertically transmitted to the next generation through seeds, influencing early plant development. While seed microbiota composition is known to be shaped by environmental, genetic, and stochastic factors, detailed insights into the factors that govern variation within and between plants of the same species remain limited. Furthermore, the dynamic of the vertically-transmitted microbes and their mode of host colonization are poorly understood. ResultsIn this study, we characterized the bacterial communities of multiple seeds originating from the same spike of adult wheat plants and demonstrated a high extent of homogeneity in the composition of microorganisms. We propagated wheat seedlings under controlled conditions to monitor host colonization by these microorganisms. Intriguingly, in spite of a relatively homogeneous composition of bacteria in individual seeds, the microbes colonizing early-stage seedlings, particularly during their initial leaf development, vary extensively, even among seedlings of seeds from the same spike. Applying a neutral assembly model, we show that the transmission of microbiomes from seeds to seedlings is primarily governed by selective processes, whereas post-transmission microbial assembly in seedlings is governed by stochastic events. ConclusionsOverall, this study introduces a novel experimental approach for investigating microbiome inheritance in plants. By leveraging high replication of seeds from the same wheat plant and field, we provide new insights into the stochastic nature of seed-associated microbiome composition and transmission in wheat.

microbiology↗

bioRxiv2024

Interspecies hybridization as a route of accessory chromosome origin in fungal species infecting wild grasses

Many fungal plant pathogens have dynamic genomic architectures that can contribute to rapid evolution and adaptation to new niches. Zymoseptoria tritici, an important fungal pathogen of wheat, has a compartmentalized and rapidly evolving genome. In the genome of the reference isolate Z. tritici IPO323, 8 of the 21 chromosomes are accessory. In spite of the profound impact on genome organization, the origin of accessory chromosomes in Z. tritici is still poorly understood. Combining genomics, transcriptomics and epigenomics, we discovered a new chromosome in Z. tritici isolates infecting wild grasses from the genus Aegilops, and we use this discovery to study the origin of accessory chromosomes. The newly identified chromosome presents similar characteristics to known accessory chromosomes in Zymoseptoria species, including presence-absence variation, low gene expression in vitro and in planta, and enrichment with heterochromatin-associated histone methylation marks (H3K27me3). Interestingly, we found an orthologous chromosome in Zymoseptoria ardabiliae, a closely related fungal species also infecting wild grasses. This ortholog chromosome also presents accessory chromosomes characteristics, but lacks the enrichment of heterochromatin-associated methylation marks. Transcriptomic analyses revealed that the orthologous chromosome in Z. ardabiliae harbors active transposable elements (TEs) congruent with lower signatures of host-genome defense mechanisms against TE expansion and spread (quantified as repeat-induced-point (RIP) mutation signatures). Our findings suggest that the chromosome has been exchanged between Z. tritici and Z. ardabiliae by introgressive hybridization events underlining the relevance of hybridization in the evolution of new accessory chromosomes. We speculate that the regulation of TEs has not yet occurred on this new accessory chromosome in Z. ardabiliae, contributing to its rapid evolution.

evolutionary biology↗

bioRxiv2024

Fitness effects of killer virus infection on wild Saccharomyces paradoxus

Endosymbioses, the intimate relationships between smaller symbionts and larger hosts, have profound impacts on eukaryotic organisms. However, symbiont effects on host fitness in natural conditions are difficult to study, especially for microbial hosts. We used killer viruses and the wild yeast Saccharomyces paradoxus to study a symbiotic viruss effect on its hosts fitness in oak litter. We cured hosts of naturally-occurring killer viruses and compared killer and cured individuals fitnesses in laboratory medium and oak litter using a unique field chamber design to house competing S. paradoxus. In the laboratory, the impact of virus loss on host fitness could be positive, negative, or neutral, depending on host identity. Trends in the forest were similar to those in the lab, although only overall strain fitness differences were significant and curing impacts differed between the forest and laboratory. These results demonstrate the importance of incorporating environmental context into studies of host-symbiont interactions.

evolutionary biology↗

bioRxiv2024

Population genomics of Macrophomina spp. reveals cryptic host specialization and evidence for meiotic recombination

Knowledge of the factors structuring populations of pathogenic fungi is fundamental to disease management efforts and basic biology. High-quality short-read sequence data were obtained for 463 Macrophomina spp. isolates collected from 91 host plant species and soil in 23 countries. Analyses revealed high diversity, admixture, and equal mating type ratios suggesting on-going recombination. Although most tested isolates could asymptomatically colonize strawberry, only isolates from a single phylogroup caused disease. In addition to strawberry, evidence for host specialization was discovered for soybean, demonstrating this broad host range pathogen contains phylogroups with cryptic specialization. Geography x isolate genotype associations were weak, suggesting these species were frequently trafficked between regions. Re-analysis using genomic data supported current species boundaries, and new molecular markers were designed to specifically identify each species. Contrary to expectations, M. phaseolina should be considered a species with both specialist and generalist populations for which meiosis can increase genetic diversity.

genomics↗

bioRxiv2024

Emergence of sympatric host-specific lineages of the fungal plant pathogen Zymoseptoria passerinii in natural ecosystems

O_LIThe barley disease Septoria Speckled Leaf Blotch, caused by the fungal pathogen Zymoseptoria passerinii, had its last outbreak in North America in the early 2000s. Although rare in agricultural settings, field sampling of wild grasses in the Middle East revealed the disease persistence in wild barley. C_LIO_LIIdentification of Z. passerinii in distinct wild barley species led us to investigate signatures of host specialization using genomics to address the mode of emergence by host tracking or host range expansion. Furthermore, we applied virulence assays and confocal laser microscopy to evaluate if the disease development differs between wild and domesticated barley. C_LIO_LIWild- and domesticated-host infecting populations have diverged, and phylogenetic relationships support the emergence of sympatric host-specific lineages. Cross-virulence assays showed that Zymoseptoria passerinii from domesticated hosts infect domesticated barley and its wild ancestor, Hordeum spontaneum. However, wild isolates from Iran did not infect domesticated barley. Wild and domesticated pathosystems have similar disease timing and progression, suggesting its persistence in natural ecosystems might be tied to environmental conditions. C_LIO_LIThe study supports that a wide range of hosts can foster the emergence of host-specific lineages in sympatry and provide novel insights into the evolution of understudied fungal pathogens on wild crop relatives. C_LI

evolutionary biology↗

bioRxiv2024

An array of Zymoseptoria tritici effectors suppress plant immune responses

Zymoseptoria tritici is the most economically significant fungal pathogen of wheat in Europe. However, despite the importance of this pathogen, the molecular interactions between pathogen and host during infection are not well understood. Herein, we describe the use of two libraries of cloned Z. tritici effectors that were screened to identify effector candidates with putative pathogen associated molecular pattern (PAMP) triggered immunity (PTI)-suppressing activity. The effectors from each library were transiently expressed in Nicotiana benthamiana, and expressing leaves were treated with bacterial or fungal PAMPs to assess the effectors ability to suppress reactive oxygen species (ROS) production. From these screens, numerous effectors were identified with PTI-suppressing activity. In addition, some effectors were able to suppress cell death responses induced by other Z. tritici secreted proteins. We used structural prediction tools to predict the putative structures of all of the Z. tritici effectors, and used these predictions to examine whether there was enrichment of specific structural signatures among the PTI-suppressing effectors. From among the libraries, multiple members of the killer protein-like 4 (KP4) and killer protein-like 6 (KP6) effector families were identified as PTI-suppressors. This observation is intriguing, as these protein families were previously associated with antimicrobial activity rather than virulence or host manipulation. This data provides mechanistic insight into immune suppression by Z. tritici during infection, and suggests that similar to biotrophic pathogens, this fungus relies on a battery of secreted effectors to suppress host immunity during early phases of colonisation.

plant biology↗

bioRxiv2023

Unleashing the secrets of plant-fungal interactions using a transformation-free confocal staining technique that supports AI-assisted quantitative analysis

Laser scanning confocal microscopys ability to generate high-contrast 3D images has become essential to studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, GFP/RFP-fusion proteins, and fluorescent labelling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins have several pitfalls including variability of expression in planta and the requirement of gene transformation. Here we used the unlabeled pathogens Parastagonospora nodorum, Pyrenophora teres f. teres, and Cercospora beticola infecting wheat, barley, and sugar beet respectively, to show the utility of a staining and imaging technique that uses propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. This method relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability. This permeability allows the staining solution to penetrate and efficiently bind to its targets, resulting in a consistent visualization of cellular structures. We have also used this staining technique in conjunction with machine learning to analyze fungal volume, which indicates the fitness of the pathogen in planta, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD). This technique is simple to use, robust, consistent across host species, and can be applied to any plant-fungal interaction. Therefore, this technique can be used to characterize model systems as well as non-model interactions where transformation is not routine.

plant biology↗

bioRxiv2023

Genome-enabled analysis of population dynamics and virulence associated loci in the oat crown rust fungus Puccinia coronata f. sp. avenae

Puccinia coronata f. sp. avenae (Pca) is an important fungal pathogen causing crown rust that impacts oat production worldwide. Genetic resistance for crop protection against Pca is often overcome by the rapid virulence evolution of the pathogen. This study investigated the factors shaping adaptive evolution of Pca using pathogen populations from distinct geographic regions within the USA and South Africa (SA). Phenotypic and genome-wide sequencing data of these diverse Pca collections, including 217 isolates, uncovered phylogenetic relationships and established distinct genetic composition between populations from northern and southern regions from the USA and SA. The population dynamics of Pca involve a bidirectional movement of inoculum between northern and southern regions of the USA and contributions from clonality and sexuality. The population from SA is solely clonal. A genome-wide association study (GWAS) employing a haplotype-resolved Pca reference genome was used to define eleven virulence-associated loci corresponding to twenty-five oat differential lines. These regions were screened to determine candidate Avr effector genes. Overall, the GWAS results allowed us to identify the underlying genetic traits controlling pathogen recognition in an oat differential set used in the USA to assign pathogen races (pathotypes). Key GWAS findings support complex genetic interactions in several oat lines suggesting allelism among resistance genes or redundancy of genes included in the differential set, multiple resistance genes recognising genetically linked Avr effector genes, or potentially epistatic relationships. A careful evaluation of the composition of the oat differential set accompanied by the development or implementation of molecular markers is recommended.

genomics↗

bioRxiv2023

Frequent horizontal chromosome transfer between asexual fungal insect pathogens

Entire chromosomes are typically only transmitted vertically from one generation to the next. The horizontal transfer of such chromosomes has long been considered improbable, yet gained recent support in several pathogenic fungi where it may affect the fitness or host specificity. To date, it is unknown how these transfers occur, how common they are and whether they can occur between different species. In this study, we show multiple independent instances of horizontal transfers of the same accessory chromosome between two distinct strains of the asexual entomopathogenic fungus Metarhizium robertsii during experimental co-infection of its insect host, the Argentine ant. Notably, only the one chromosome - but no other - was transferred from the donor to the recipient strain. The recipient strain, now harboring the accessory chromosome, exhibited a competitive advantage under certain host conditions. By phylogenetic analysis we further demonstrate that the same accessory chromosome was horizontally transferred in a natural environment between M. robertsii and another congeneric insect pathogen, M. guizhouense. Hence horizontal chromosome transfer is not limited to the observed frequent events within species during experimental infections but also occurs naturally across species. The transferred accessory chromosome contains genes that might be involved in its preferential horizontal transfer, encoding putative histones and histone-modifying enzymes, but also putative virulence factors that may support its establishment. Our study reveals that both intra- and interspecies horizontal transfer of entire chromosomes is more frequent than previously assumed, likely representing a not uncommon mechanism for gene exchange. Significance StatementThe enormous success of bacterial pathogens has been attributed to their ability to exchange genetic material between one another. Similarly, in eukaryotes, horizontal transfer of genetic material allowed the spread of virulence factors across species. The horizontal transfer of whole chromosomes could be an important pathway for such exchange of genetic material, but little is known about the origin of transferable chromosomes and how frequently they are exchanged. Here, we show that the transfer of accessory chromosomes - chromosomes that are non-essential but may provide fitness benefits - is common during fungal co-infections and is even possible between distant pathogenic species, highlighting the importance of horizontal gene transfer via chromosome transfer also for the evolution and function of eukaryotic pathogens.

genomics↗

bioRxiv2023

Large-scale analyses reveal the contribution of adaptive evolution in pathogenic and non-pathogenic fungal species

Genome studies of fungal pathogens have presented evidence for exceptionally high rates of evolution. It has been proposed that rapid adaptation is a hallmark of pathogen evolution that facilitates the invasion of new host niches and the overcoming of intervention strategies such as fungicide applications and drug treatments. To which extent high levels of genetic variation within and between species correlate with adaptive protein evolution in fungi more generally has so far not been explored. In this study, we addressed the contribution of adaptive evolution relative to genetic drift in 20 fungal species, hereby exploring genetic variation in 2,478 fungal genomes. We reannotated positions of protein-coding genes to obtain a high-quality dataset of 234,427 full-length core gene and 25,612 accessory gene alignments. We applied an extension of the McDonald-Kreitman test that models the distributions of fitness effects to infer the rate of adaptive ({omega}A) and non-adaptive ({omega}NA) non-synonymous substitutions in protein-coding genes. To explore the relevance of recombination on local adaptation rates, we inferred the population genomic recombination rate for all 20 species. Our analyses reveal extensive variation in rates of adaptation and show that high rates of adaptation are not a hallmark of a pathogenic lifestyle. Up to 83% of non-synonymous substitutions are adaptive in the species Parastagonospora nodorum. However, non-synonymous substitutions in other species, including the prominent rice-infecting pathogen Magnaporthe oryzae, are predominantly non-adaptive (neutral or slightly deleterious). Correlating adaptation measures with effective population size and recombination rate, we show that effective population size is a primary determinant of adaptive evolution in fungi. At the genome scale, recombination rate variation explains variation in both {omega}A and {omega}NA. Finally, we demonstrate the robustness of our estimates using simulations. We underline the value of population genetic principles in studies of fungal evolution, and we highlight the importance of demographic processes in adaptive evolution of pathogenic and non-pathogenic species.

evolutionary biology↗

bioRxiv2023

Emergence and spread of the barley net blotch pathogen coincided with crop domestication and cultivation history

Fungal pathogens cause devastating disease in crops. Understanding the evolutionary origin of pathogens is essential to the prediction of future disease emergence and the potential of pathogens to disperse. The fungus Pyrenophora teres f. teres causes net form net blotch (NFNB), an economically significant disease of barley. In this study, we have used 104 P. teres f. teres genomes from four continents to explore the population structure and demographic history of the fungal pathogen. We showed that P. teres f. teres is structured into populations that tend to be geographically restricted to different regions. Using Multiple Sequentially Markovian Coalescent and machine learning approaches we demonstrated that the demographic history of the pathogen correlates with the history of barley, highlighting the importance of human migration and trade in spreading the pathogen. Exploring signatures of natural selection, we identified several population-specific selective sweeps that colocalized with genomic regions enriched in putative virulence genes, and loci previously identified as determinants of virulence specificities by quantitative trait locus analyses. This reflects rapid adaptation to local hosts and environmental conditions of P. teres f. teres as it spread with barley. Our research highlights how human activities can contribute to the spread of pathogens that significantly impact the productivity of field crops.

evolutionary biology↗

bioRxiv2023

The apoplastic space of two wheat genotypes provide highly different environment for pathogen colonization: Insights from proteome and microbiome profiling

The intercellular space comprising the plant apoplast harbors a diverse range of microorganisms. The apoplastic interface represents the main compartment for interactions between proteins produced and secreted by the plant and the microbial endophytes. The outcomes of these interactions can play a role in plant cell wall metabolism, stress tolerance, and plant-pathogen resistance. So far the underlying factors that determine microbiota composition in the apoplast are not fully understood. However, it is considered that cell wall composition, nutrient availability, and the plant immune system are main determinants of microbiota composition. The plant immune system is considered to play a crucial role in modulating microbiota composition through the recognition of specific microbe-associated molecular patterns and the activation of defense responses. Hereby the plant may restrict non-beneficial microbial members and facilitate the propagation of beneficial ones. In this study, we investigated changes in the apoplastic environment during pathogen invasion using wheat as a model system. Infection of wheat with Zymoseptoria tritici, a fungal pathogen, resulted in notable alterations in the apoplast composition, reduced microbial diversity, and the accumulation of antimicrobial defense metabolites. Intriguingly, certain core microbial members persisted even in the presence of pathogen-induced immune responses, indicating their ability to evade or tolerate host immune defenses. To further explore these dynamics, we developed a protocol for extracting apoplastic fluids from wheat leaves and conducted proteome analyses to characterize the dynamic environment of the wheat leaves. Our findings uncovered a highly variable apoplastic environment that selects for microbes with specific adaptations. Notably, a core microbial community enriched in the resistant wheat cultivar exhibited antagonistic activity against Z. tritici, suggesting a potential role in conferring pathogen defense. This study advances our understanding of the dynamic interactions and adaptations of the wheat apoplastic microbiota during pathogen invasion, emphasizing the pivotal role of microbial interactions in pathogen defenses.

microbiology↗

bioRxiv2022

Repeat-induced point mutation and gene conversion coinciding with heterochromatin shape the genome of a plant pathogenic fungus

Meiosis is associated with genetic changes in the genome - via recombination, gene conversion, and mutations. The occurrence of gene conversion and mutations during meiosis may further be influenced by the chromatin conformation, in analogy to what is known for mutations during mitosis. To date, however, the exact distribution and type of meiosis-associated changes and the role of the chromatin conformation in this context is largely unexplored. Here, we determine recombination, gene conversion, and de novo mutations using whole-genome sequencing of all meiotic products of 23 individual meioses in Zymoseptoria tritici, an important pathogen of wheat. We could confirm a high genome-wide recombination rate of 65 cM/Mb and see higher recombination rates on the accessory compared to core chromosomes. A substantial fraction of 0.16% of all polymorphic markers was affected by gene conversions, showing a weak GC-bias, and occurring at higher frequency in regions of constitutive heterochromatin, indicated by the histone modification H3K9me3. The de novo mutation rate associated with meiosis was approx. three orders of magnitude higher than the corresponding mitotic mutation rate. Importantly, repeat-induced point mutation (RIP), a fungal defense mechanism against duplicated sequences, is active in Z. tritici and responsible for the majority of these de novo meiotic mutations. Our results indicate that the genetic changes associated with meiosis are a major source of variability in the genome of an important plant pathogen and shape its evolutionary trajectory. ImportanceThe impact of meiosis on the genome composition via gene conversion and mutations is mostly poorly understood, in particular for non-model species. Here, we sequenced all four meiotic products for 23 individual meioses and determined the genetic changes caused by meiosis for the important fungal wheat pathogen Zymoseptoria tritici. We found a high rate of gene conversions and an effect of the chromatin conformation on gene conversion rates. Higher conversion rates were found in regions enriched with the H3K9me3 - a mark for constitutive heterochromatin. Most importantly, meiosis was associated with a much higher frequency of de novo mutations than mitosis. 78% of the meiotic mutations were caused by repeat-induced point mutations - a fungal defense mechanism against duplicated sequences. In conclusion, the genetic changes associated with meiosis are therefore a major factor shaping the genome of this fungal pathogen.

genomics↗

bioRxiv2021

Differential regulation and production of secondary metabolites among isolates of the fungal wheat pathogen Zymoseptoria tritici

The genome of the wheat pathogenic fungus, Zymoseptoria tritici, represents extensive presence-absence variation in gene content. Here, we addressed variation in biosynthetic gene clusters (BGCs) content and biochemical profiles among three isolates. We analysed secondary metabolite properties based on genome, transcriptome and metabolome data. The isolates represent highly distinct genome architecture, but harbor similar repertoire of BGCs. Expression profiles for most BGCs show comparable patterns of regulation among the isolates, suggesting a conserved "biochemical infection program". For all three isolates, we observed a strong up-regulation of an abscisic acid (ABA) gene cluster during biotrophic host colonization, indicating that Z. tritici potentially interfere with host defenses by the biosynthesis of this phytohormone. Further, during in vitro growth the isolates show similar metabolomes congruent with the predicted BGC content. We assessed if secondary metabolite production is regulated by histone methylation using a mutant impaired in formation of facultative heterochromatin (H3K27me3). In contrast to other ascomycete fungi, chromatin modifications play a less prominent role in regulation of secondary metabolites. In summary, we show that Z. tritici has a conserved program of secondary metabolite production contrasting the immense variation in effector expression, some of these metabolites might play a key role during host colonization. Originality-Significance StatementZymoseptoria tritici is one of the most devastating pathogens of wheat. So far the molecular determinants of virulence and their regulation are poorly understood. Previous studies have focused on proteinasous virulence factors and their extensive diversity. In this study, we focus on secondary metabolites produced by Z. tritici. Using a comparative framework, we here characterize core and non-core metabolites produced by Z. tritici by combining genome, transcriptome and metabolome datasets. Our findings indicate highly conserved biochemical profiles contrasting genetic and phenotypic diversity of the field isolates investigated here. This discovery has relevance for future crop protection strategies.

microbiology↗

bioRxiv2020

Genome-wide association studies reveal the complex genetic architecture of DMI fungicide resistance in Cercospora beticola

Cercospora leaf spot is the most important disease of sugar beet worldwide. The disease is caused by the fungus Cercospora beticola and is managed principally by timely application of fungicides including those of the sterol demethylation inhibitor (DMI) class. However, reliance on DMIs has caused an increase in resistance to this class of fungicides in multiple C. beticola populations. To better understand the genetic and evolutionary basis for resistance in C. beticola, a genome-wide association study (GWAS) and selective sweep analysis were conducted for the first time in this fungal plant pathogen. We performed whole genome resequencing of 190 C. beticola isolates predominantly from North Dakota and Minnesota that were phenotyped for sensitivity to tetraconazole, the most widely used DMI fungicide in this region. GWAS identified mutations in genes associated with DMI fungicide resistance including a Regulator of G-protein Signaling (RGS) protein, an ATP-binding cassette (ABC) pleiotropic drug resistance transporter, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK), and a gene annotated as a hypothetical protein. A SNP upstream of CbCYP51, the gene encoding the target of DMI fungicides, was also identified via GWAS. Haplotype analysis of CbCYP51 identified a synonymous mutation (E170) in high linkage disequilibrium with the upstream SNP, and multiple non-synonymous mutations (L144F, I387M and Y464S) associated with DMI resistance. Additionally, a putative codon bias effect for the L144F substitution was identified that generated different resistance potentials. We also identified a CbCYP51 paralog in C. beticola, CbCYP51-like, with high protein homology to CYP51C found uniquely in Fusarium species but CbCYP51-like does not appear to influence DMI sensitivity. Genome-wide scans of selection showed that several of the GWAS mutations for fungicide resistance resided in regions that have recently undergone a selective sweep. Using radial plate growth on selected media as a fitness proxy, we did not find a trade-off associated with DMI fungicide resistance suggesting that resistance mutations can persist in C. beticola populations. Taken together, we show that population genomic data from a crop pathogen can allow the identification of mutations conferring fungicide resistance and inform about their origins in the pathogen population.

evolutionary biology↗