Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance.

Correction: Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

[This corrects the article DOI: 10.1371/journal.pbio.3002052.].

Recent reactivation of a pathogenicity-associated transposable element is associated with major chromosomal rearrangements in a fungal wheat pathogen.

Transposable elements (TEs) are key drivers of genomic variation contributing to recent adaptation in most species. Yet, the evolutionary origins and insertion dynamics within species remain poorly understood. We recapitulate the spread of the pathogenicity-associated Styx element across five species that last diverged ∼11 000 years ago. We show that the element likely originated in the Zymoseptoria fungal pathogen genus and underwent multiple independent reactivation events. Using a global 900-genome panel of the wheat pathogen Zymoseptoria tritici, we assess Styx copy number variation and identify renewed transposition activity in Oceania and South America. We show that the element can mobilize to create additional Styx copies in a four-generation pedigree. Importantly, we find that new copies of the element are not affected by genomic defenses suggesting minimal control against the element. Styx copies are preferentially located in recombination breakpoints and likely triggered multiple types of large chromosomal rearrangements. Taken together, we establish the origin, diversification and reactivation of a highly active TE with likely major consequences for chromosomal integrity and the expression of disease.

Genomic Signatures of Domestication in a Fungus Obligately Farmed by Leafcutter Ants.

The naturally selected fungal crop (Leucoagaricus gongylophorus) farmed by leafcutter ants shows striking parallels with artificially selected plant crops domesticated by humans (e.g. polyploidy, engorged nutritional rewards, and dependence on cultivation). To date, poorly resolved L. gongylophorus genome assemblies based on short-read sequencing have constrained hypotheses about how millions of years under cultivation by ants shaped the fungal crop genome and potentially drove domestication. We use PacBio HiFi sequencing of L. gongylophorus from the leafcutter ant Atta colombica to identify 18 putatively novel biosynthetic gene clusters that likely cemented life as a cultivar (e.g. plant fragment degradation, ant-farmer communication, and antimicrobial defense). Comparative analyses with cultivated and free-living fungi showed genomic signatures of stepwise domestication transitions: (i) free-living to ant-cultivated: loss of genes conferring stress response and detoxification; (ii) hyphal food to engorged nutritional rewards: expansions of genes governing cellular homeostasis, carbohydrate metabolism, and siderophore biosynthesis; and (iii) detrital provisioning to freshly cut plant fragments: gene expansions promoting cell wall biosynthesis, fatty acid metabolism, and DNA repair. Comparisons across L. gongylophorus fungi farmed by 3 leafcutter ant species highlight genomic signatures of exclusively vertical clonal propagation and widespread transposable element activity. These results show how natural selection can shape domesticated cultivar genomes toward long-term ecological resilience of farming systems that have thrived across millennia.

A devil's bargain with transposable elements in plant pathogens.

Transposable elements (TEs) spread in genomes through self-copying mechanisms and are a major cause of genome expansions. Plant pathogens have finely tuned the expression of virulence factors to rely on epigenetic control targeted at nearby TEs. Stress experienced during the plant infection process leads to derepression of TEs and concurrently allows the expression of virulence factors. We argue that the derepression of TEs elements causes an evolutionary conflict by favoring TEs that can be reactivated. Active TEs and recent genome size expansions indicate that plant pathogens could face long-term consequences from the short-term benefit of fine-tuning the infection process. Hence, encoding key virulence factors close to TEs under epigenetic control constitutes a devil's bargain for pathogens.

Unraveling coevolutionary dynamics using ecological genomics.

Coevolutionary interactions, from the delicate co-dependency in mutualistic interactions to the antagonistic relationship of hosts and parasites, are a ubiquitous driver of adaptation. Surprisingly, little is known about the genomic processes underlying coevolution in an ecological context. However, species comprise genetically differentiated populations that interact with temporally variable abiotic and biotic environments. We discuss the recent advances in coevolutionary theory and genomics as well as shortcomings, to identify coevolving genes that take into account this spatial and temporal variability of coevolution, and propose a practical guide to understand the dynamic of coevolution using an ecological genomics lens.

Recent transposable element bursts are associated with the proximity to genes in a fungal plant pathogen.

The activity of transposable elements (TEs) contributes significantly to pathogen genome evolution. TEs often destabilize genome integrity but may also confer adaptive variation in pathogenicity or resistance traits. De-repression of epigenetically silenced TEs often initiates bursts of transposition activity that may be counteracted by purifying selection and genome defenses. However, how these forces interact to determine the expansion routes of TEs within a pathogen species remains largely unknown. Here, we analyzed a set of 19 telomere-to-telomere genomes of the fungal wheat pathogen Zymoseptoria tritici. Phylogenetic reconstruction and ancestral state estimates of individual TE families revealed that TEs have undergone distinct activation and repression periods resulting in highly uneven copy numbers between genomes of the same species. Most TEs are clustered in gene poor niches, indicating strong purifying selection against insertions near coding sequences, or as a consequence of insertion site preferences. TE families with high copy numbers have low sequence divergence and strong signatures of defense mechanisms (i.e., RIP). In contrast, small non-autonomous TEs (i.e., MITEs) are less impacted by defense mechanisms and are often located in close proximity to genes. Individual TE families have experienced multiple distinct burst events that generated many nearly identical copies. We found that a Copia element burst was initiated from recent copies inserted substantially closer to genes compared to older copies. Overall, TE bursts tended to initiate from copies in GC-rich niches that escaped inactivation by genomic defenses. Our work shows how specific genomic environments features provide triggers for TE proliferation in pathogen genomes.

Combined reference-free and multi-reference based GWAS uncover cryptic variation underlying rapid adaptation in a fungal plant pathogen.

Microbial pathogens often harbor substantial functional diversity driven by structural genetic variation. Rapid adaptation from such standing variation threatens global food security and human health. Genome-wide association studies (GWAS) provide a powerful approach to identify genetic variants underlying recent pathogen adaptation. However, the reliance on single reference genomes and single nucleotide polymorphisms (SNPs) obscures the true extent of adaptive genetic variation. Here, we show quantitatively how a combination of multiple reference genomes and reference-free approaches captures substantially more relevant genetic variation compared to single reference mapping. We performed reference-genome based association mapping across 19 reference-quality genomes covering the diversity of the species. We contrasted the results with a reference-free (i.e., k-mer) approach using raw whole-genome sequencing data in a panel of 145 strains collected across the global distribution range of the fungal wheat pathogen Zymoseptoria tritici. We mapped the genetic architecture of 49 life history traits including virulence, reproduction and growth in multiple stressful environments. The inclusion of additional reference genome SNP datasets provides a nearly linear increase in additional loci mapped through GWAS. Variants detected through the k-mer approach explained a higher proportion of phenotypic variation than a reference genome-based approach and revealed functionally confirmed loci that classic GWAS approaches failed to map. The power of GWAS in microbial pathogens can be significantly enhanced by comprehensively capturing structural genetic variation. Our approach is generalizable to a large number of species and will uncover novel mechanisms driving rapid adaptation of pathogens.

A secreted protease-like protein in Zymoseptoria tritici is responsible for avirulence on Stb9 resistance gene in wheat.

Zymoseptoria tritici is the fungal pathogen responsible for Septoria tritici blotch on wheat. Disease outcome in this pathosystem is partly determined by isolate-specific resistance, where wheat resistance genes recognize specific fungal factors triggering an immune response. Despite the large number of known wheat resistance genes, fungal molecular determinants involved in such cultivar-specific resistance remain largely unknown. We identified the avirulence factor AvrStb9 using association mapping and functional validation approaches. Pathotyping AvrStb9 transgenic strains on Stb9 cultivars, near isogenic lines and wheat mapping populations, showed that AvrStb9 interacts with Stb9 resistance gene, triggering an immune response. AvrStb9 encodes an unusually large avirulence gene with a predicted secretion signal and a protease domain. It belongs to a S41 protease family conserved across different filamentous fungi in the Ascomycota class and may constitute a core effector. AvrStb9 is also conserved among a global Z. tritici population and carries multiple amino acid substitutions caused by strong positive diversifying selection. These results demonstrate the contribution of an 'atypical' conserved effector protein to fungal avirulence and the role of sequence diversification in the escape of host recognition, adding to our understanding of host-pathogen interactions and the evolutionary processes underlying pathogen adaptation.

Variability in an effector gene promoter of a necrotrophic fungal pathogen dictates epistasis and effector-triggered susceptibility in wheat.

The fungus Parastagonospora nodorum uses proteinaceous necrotrophic effectors (NEs) to induce tissue necrosis on wheat leaves during infection, leading to the symptoms of septoria nodorum blotch (SNB). The NEs Tox1 and Tox3 induce necrosis on wheat possessing the dominant susceptibility genes Snn1 and Snn3B1/Snn3D1, respectively. We previously observed that Tox1 is epistatic to the expression of Tox3 and a quantitative trait locus (QTL) on chromosome 2A that contributes to SNB resistance/susceptibility. The expression of Tox1 is significantly higher in the Australian strain SN15 compared to the American strain SN4. Inspection of the Tox1 promoter region revealed a 401 bp promoter genetic element in SN4 positioned 267 bp upstream of the start codon that is absent in SN15, called PE401. Analysis of the world-wide P. nodorum population revealed that a high proportion of Northern Hemisphere isolates possess PE401 whereas the opposite was observed in representative P. nodorum isolates from Australia and South Africa. The presence of PE401 removed the epistatic effect of Tox1 on the contribution of the SNB 2A QTL but not Tox3. PE401 was introduced into the Tox1 promoter regulatory region in SN15 to test for direct regulatory roles. Tox1 expression was markedly reduced in the presence of PE401. This suggests a repressor molecule(s) binds PE401 and inhibits Tox1 transcription. Infection assays also demonstrated that P. nodorum which lacks PE401 is more pathogenic on Snn1 wheat varieties than P. nodorum carrying PE401. An infection competition assay between P. nodorum isogenic strains with and without PE401 indicated that the higher Tox1-expressing strain rescued the reduced virulence of the lower Tox1-expressing strain on Snn1 wheat. Our study demonstrated that Tox1 exhibits both 'selfish' and 'altruistic' characteristics. This offers an insight into a complex NE-NE interaction that is occurring within the P. nodorum population. The importance of PE401 in breeding for SNB resistance in wheat is discussed.

Two-speed genomes of Epichloe fungal pathogens show contrasting signatures of selection between species and across populations.

Antagonistic selection between pathogens and their hosts can drive rapid evolutionary change and leave distinct molecular footprints of past and ongoing selection in the genomes of the interacting species. Despite an increasing availability of tools able to identify signatures of selection, the genetic mechanisms underlying coevolutionary interactions and the specific genes involved are still poorly understood, especially in heterogeneous natural environments. We searched the genomes of two species of Epichloe plant pathogen for evidence of recent selection. The Epichloe genus includes highly host-specific species that can sterilize their grass hosts. We performed selection scans using genome-wide SNP data from seven natural populations of two co-occurring Epichloe sibling species specialized on different hosts. We found evidence of recent (and ongoing) selective sweeps across the genome in both species. However, selective sweeps were more abundant in the species with a larger effective population size. Sweep regions often overlapped with highly polymorphic AT-rich regions supporting the role of these genome compartments in adaptive evolution. Although most loci under selection were specific to individual populations, we could also identify several candidate genes targeted by selection in sweep regions shared among populations. The genes encoded small secreted proteins typical of fungal effectors and cell wall-degrading enzymes. By investigating the genomic signatures of selection across multiple populations and species, this study contributes to our understanding of complex adaptive processes in natural plant pathogen systems.

Genome-wide expression QTL mapping reveals the highly dynamic regulatory landscape of a major wheat pathogen.

BACKGROUND: In agricultural ecosystems, outbreaks of diseases are frequent and pose a significant threat to food security. A successful pathogen undergoes a complex and well-timed sequence of regulatory changes to avoid detection by the host immune system; hence, well-tuned gene regulation is essential for survival. However, the extent to which the regulatory polymorphisms in a pathogen population provide an adaptive advantage is poorly understood. RESULTS: We used Zymoseptoria tritici, one of the most important pathogens of wheat, to generate a genome-wide map of regulatory polymorphism governing gene expression. We investigated genome-wide transcription levels of 146 strains grown under nutrient starvation and performed expression quantitative trait loci (eQTL) mapping. We identified cis-eQTLs for 65.3% of all genes and the majority of all eQTL loci are within 2kb upstream and downstream of the transcription start site (TSS). We also show that polymorphism in different gene elements contributes disproportionally to gene expression variation. Investigating regulatory polymorphism in gene categories, we found an enrichment of regulatory variants for genes predicted to be important for fungal pathogenesis but with comparatively low effect size, suggesting a separate layer of gene regulation involving epigenetics. We also show that previously reported trait-associated SNPs in pathogen populations are frequently cis-regulatory variants of neighboring genes with implications for the trait architecture. CONCLUSIONS: Overall, our study provides extensive evidence that single populations segregate large-scale regulatory variation and are likely to fuel rapid adaptation to resistant hosts and environmental change.

Histone H3K27 Methylation Perturbs Transcriptional Robustness and Underpins Dispensability of Highly Conserved Genes in Fungi.

Epigenetic modifications are key regulators of gene expression and underpin genome integrity. Yet, how epigenetic changes affect the evolution and transcriptional robustness of genes remains largely unknown. Here, we show how the repressive histone mark H3K27me3 underpins the trajectory of highly conserved genes in fungi. We first performed transcriptomic profiling on closely related species of the plant pathogen Fusarium graminearum species complex. We determined transcriptional responsiveness of genes across environmental conditions to determine expression robustness. To infer evolutionary conservation, we used a framework of 23 species across the Fusarium genus including three species covered with histone methylation data. Gene expression variation is negatively correlated with gene conservation confirming that highly conserved genes show higher expression robustness. In contrast, genes marked by H3K27me3 do not show such associations. Furthermore, highly conserved genes marked by H3K27me3 encode smaller proteins, exhibit weaker codon usage bias, higher levels of hydrophobicity, show lower intrinsically disordered regions, and are enriched for functions related to regulation and membrane transport. The evolutionary age of conserved genes with H3K27me3 histone marks falls typically within the origins of the Fusarium genus. We show that highly conserved genes marked by H3K27me3 are more likely to be dispensable for survival during host infection. Lastly, we show that conserved genes exposed to repressive H3K27me3 marks across distantly related Fusarium fungi are associated with transcriptional perturbation at the microevolutionary scale. In conclusion, we show how repressive histone marks are entangled in the evolutionary fate of highly conserved genes across evolutionary timescales.

The population genetics of adaptation through copy number variation in a fungal plant pathogen.

Microbial pathogens can adapt rapidly to changing environments such as the application of pesticides or host resistance. Copy number variations (CNVs) are a major source of adaptive genetic variation for recent adaptation. Here, we analyse how a major fungal pathogen of barley, Rhynchosporium commune, has adapted to the host environment and fungicide applications. We screen the genomes of 125 isolates sampled across a worldwide set of populations and identify a total of 7,879 gene duplications and 116 gene deletions. Most gene duplications result from segmental chromosomal duplications. Although CNVs are generally under negative selection, we find that genes affected by CNVs are enriched in functions related to host exploitation (i.e., effectors and cell-wall-degrading enzymes). We perform genome-wide association studies (GWAS) and identify a large segmental duplication of CYP51A that has contributed to the emergence of azole resistance and a duplication encompassing an effector gene affecting virulence. We show that the adaptive CNVs were probably created by recently active transposable element families. Moreover, we find that specific transposable element families are important drivers of recent gene CNV. Finally, we use a genome-wide single nucleotide polymorphism data set to replicate the GWAS and contrast it with the CNV-focused analysis. Together, our findings show how extensive segmental duplications create the raw material for recent adaptation in global populations of a fungal pathogen.

Virulence Associations and Global Context of AvrStb6 Genetic Diversity in Iranian Populations of Zymoseptoria tritici.

Managing pathogen damage in wheat production is important for sustaining yields. Fungal plant pathogen genomes encode many small secreted proteins acting as effectors that play key roles in the successful colonization of host tissue and triggering host defenses. AvrStb6 is the first described Zymoseptoria tritici avirulence effector, which triggers Stb6-mediated immunity in the wheat host in a gene-for-gene manner. Evasion of major resistance factors such as Stb6 challenges deployment decisions on wheat cultivars. In this study, we analyzed the evolution of the AvrStb6 effector in Iranian isolates of Z. tritici. In total, 78 isolates were isolated and purified from 30 infected wheat specimens collected from the East Azerbaijan and Ardabil provinces of Iran. The pathogenicity of all isolates was evaluated on the susceptible wheat cultivar 'Tajan'. A subset of 40 isolates were also tested for pathogenicity on the resistant cultivar 'Shafir' carrying Stb6. Genetic diversity at the AvrStb6 locus was analyzed for 14 isolates covering the breadth of the observed disease severity. The AvrStb6 sequence variation was high, with virulent isolates carrying highly diverse AvrStb6 haplotypes. In an analysis including more than 1,000 additional AvrStb6 sequences from a global set of isolates, we found that virulent isolates carried AvrStb6 haplotypes either clustering with known virulent haplotypes on different continents or constituting previously unknown haplotypes. Furthermore, we found that AvrStb6 variants from avirulent isolates clustered with known avirulent genotypes from Europe. Our study highlights the relevance of AvrStb6 for Z. tritici virulence and the exceptional global diversity patterns of this effector.

Genome-wide association mapping reveals genes underlying population-level metabolome diversity in a fungal crop pathogen.

BACKGROUND: Fungi produce a wide range of specialized metabolites (SMs) involved in biotic interactions. Pathways for the production of SMs are often encoded in clusters of tightly arranged genes identified as biosynthetic gene clusters. Such gene clusters can undergo horizontal gene transfers between species and rapid evolutionary change within species. The acquisition, rearrangement, and deletion of gene clusters can generate significant metabolome diversity. However, the genetic basis underlying variation in SM production remains poorly understood. RESULTS: Here, we analyzed the metabolite production of a large population of the fungal pathogen of wheat, Zymoseptoria tritici. The pathogen causes major yield losses and shows variation in gene clusters. We performed untargeted ultra-high performance liquid chromatography-high resolution mass spectrometry to profile the metabolite diversity among 102 isolates of the same species. We found substantial variation in the abundance of the detected metabolites among isolates. Integrating whole-genome sequencing data, we performed metabolite genome-wide association mapping to identify loci underlying variation in metabolite production (i.e., metabolite-GWAS). We found that significantly associated SNPs reside mostly in coding and gene regulatory regions. Associated genes encode mainly transport and catalytic activities. The metabolite-GWAS identified also a polymorphism in the 3'UTR region of a virulence gene related to metabolite production and showing expression variation. CONCLUSIONS: Taken together, our study provides a significant resource to unravel polymorphism underlying metabolome diversity within a species. Integrating metabolome screens should be feasible for a range of different plant pathogens and help prioritize molecular studies.

A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization.

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.

Temporal changes in pathogen diversity in a perennial plant-pathogen-hyperparasite system.

Hyperparasites can affect the evolution of pathosystems by influencing the stability of both pathogen and host populations. However, how pathogens of perennial hosts evolve in the presence of a hyperparasite has rarely been studied. Here, we investigated temporal changes in genetic diversity of the invasive chestnut blight pathogen Cryphonectria parasitica in the presence of its parasitic mycovirus Cryphonectria hypovirus 1 (CHV1). The virus reduces fungal virulence and represents an effective natural biocontrol agent against chestnut blight in Europe. We analysed genome-wide diversity and CHV1 prevalence in C. parasitica populations in southern Switzerland that were sampled twice at an interval of about 30 years. Overall, we found that both pathogen population structure and CHV1 prevalence were retained over time. The results suggest that recent bottlenecks have influenced the structure of C. parasitica populations in southern Switzerland. Strong balancing selection signals were found at a single vegetative incompatibility (vic) locus, consistent with negative frequency-dependent selection imposed by the vegetative incompatibility system. High levels of mating among related individuals (i.e., inbreeding) and genetic drift are probably at the origin of imbalanced allele ratios at vic loci and subsequently low vc type diversity. Virus infection rates were stable at ~30% over the study period and we found no significant impact of the virus on fungal population diversity. Consequently, the efficacy of CHV1-mediated biocontrol was probably retained.

High-quality genome assembly of Pseudocercospora ulei the main threat to natural rubber trees.

Pseudocercospora ulei is the causal agent of South American Leaf Blight (SALB), the main disease affecting Hevea brasiliensis rubber tree, a native species to the Amazon. Rubber tree is a major crop in South American countries and SALB disease control strategies would benefit from the availability of genomic resources for the fungal pathogen. Here, we assembled and annotated the P. ulei genome. Shotgun sequencing was performed using second and third generation sequencing technologies. We present the first P. ulei high-quality genome assembly, the largest among Mycosphaerellaceae, with 93.8 Mbp, comprising 215 scaffolds, an N50 of 2.8 Mbp and a BUSCO gene completeness of 97.5%. We identified 12,745 protein-coding gene models in the P. ulei genome with 756 genes encoding secreted proteins and 113 genes encoding effector candidates. Most of the genome (80%) is composed of repetitive elements dominated by retrotransposons of the Gypsy superfamily. P. ulei has the largest genome size among Mycosphaerellaceae, with the highest TE content. In conclusion, we have established essential genomic resources for a wide range of studies on P. ulei and related species.

Rapid sequence evolution driven by transposable elements at a virulence locus in a fungal wheat pathogen.

BACKGROUND: Plant pathogens cause substantial crop losses in agriculture production and threaten food security. Plants evolved the ability to recognize virulence factors and pathogens have repeatedly escaped recognition due rapid evolutionary change at pathogen virulence loci (i.e. effector genes). The presence of transposable elements (TEs) in close physical proximity of effector genes can have important consequences for gene regulation and sequence evolution. Species-wide investigations of effector gene loci remain rare hindering our ability to predict pathogen evolvability. RESULTS: Here, we performed genome-wide association studies (GWAS) on a highly polymorphic mapping population of 120 isolates of Zymoseptoria tritici, the most damaging pathogen of wheat in Europe. We identified a major locus underlying significant variation in reproductive success of the pathogen and damage caused on the wheat cultivar Claro. The most strongly associated locus is intergenic and flanked by genes encoding a predicted effector and a serine-type endopeptidase. The center of the locus contained a highly dynamic region consisting of multiple families of TEs. Based on a large global collection of assembled genomes, we show that the virulence locus has undergone substantial recent sequence evolution. Large insertion and deletion events generated length variation between the flanking genes by a factor of seven (5-35 kb). The locus showed also strong signatures of genomic defenses against TEs (i.e. RIP) contributing to the rapid diversification of the locus. CONCLUSIONS: In conjunction, our work highlights the power of combining GWAS and population-scale genome analyses to investigate major effect loci in pathogens.