Search

Species harbor extensive structural variation underpinning recent adaptive evolution. However, the causality between genomic features and the induction of new rearrangements is poorly established. Here, we analyze a global set of telomere-to-telomere genome assemblies of a fungal pathogen of wheat to establish a nucleotide-level map of structural variation. We show that the recent emergence of pesticide resistance has been disproportionally driven by rearrangements. We use machine learning to train a model on structural variation events based on 30 chromosomal sequence features. We show that base composition and gene density are the major determinants of structural variation. Retrotransposons explain most inversion, indel and duplication events. We apply our model to Arabidopsis thaliana and show that our approach extends to more complex genomes. Finally, we analyze complete genomes of haploid offspring in a four-generation pedigree. Meiotic crossover locations are enriched for new rearrangements consistent with crossovers being mutational hotspots. The model trained on species-wide structural variation accurately predicts the position of >74% of newly generated variants along the pedigree. The predictive power highlights causality between specific sequence features and the induction of chromosomal rearrangements. Our work demonstrates that training sequence-derived models can accurately identify regions of intrinsic DNA instability in eukaryotic genomes.

Infections by more than one strain of a pathogen predominate under natural conditions. Mixed infections can have significant, though often unpredictable, consequences for overall virulence, pathogen transmission and evolution. However, effects of mixed infection on disease development in plants often remain unclear and the critical factors that determine the outcome of mixed infections remain unknown. The fungus Zymoseptoria tritici forms genetically diverse infections in wheat fields. Here, for a range of pathogen traits, we experimentally decompose the infection process to determine how the outcomes and consequences of mixed infections are mechanistically realized. Different strains of Z. tritici grow in close proximity and compete in the wheat apoplast, resulting in reductions in growth of individual strains and in pathogen reproduction. We observed different outcomes of competition at different stages of the infection. Overall, more virulent strains had higher competitive ability during host colonization, and less virulent strains had higher transmission potential. We showed that within-host competition can have a major effect on infection dynamics and pathogen population structure in a pathogen and host genotype-specific manner. Consequently, mixed infections likely have a major effect on the development of septoria tritici blotch epidemics and the evolution of virulence in Z. tritici., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Zymoseptoria tritici is the causal agent of Septoria tritici blotch, a major pathogen of wheat globally and the most damaging pathogen of wheat in Europe. A gene-for-gene (GFG) interaction between Z. tritici and wheat cultivars carrying the Stb6 resistance gene has been postulated for many years, but the genes have not been identified. We identified AvrStb6 by combining quantitative trait locus mapping in a cross between two Swiss strains with a genome-wide association study using a natural population of c. 100 strains from France. We functionally validated AvrStb6 using ectopic transformations. AvrStb6 encodes a small, cysteine-rich, secreted protein that produces an avirulence phenotype on wheat cultivars carrying the Stb6 resistance gene. We found 16 nonsynonymous single nucleotide polymorphisms among the tested strains, indicating that AvrStb6 is evolving very rapidly. AvrStb6 is located in a highly polymorphic subtelomeric region and is surrounded by transposable elements, which may facilitate its rapid evolution to overcome Stb6 resistance. AvrStb6 is the first avirulence gene to be functionally validated in Z. tritici, contributing to our understanding of avirulence in apoplastic pathogens and the mechanisms underlying GFG interactions between Z. tritici and wheat., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Zymoseptoria tritici is an ascomycete fungus that causes Septoria tritici blotch, a globally distributed foliar disease on wheat. Z. tritici populations are highly polymorphic and exhibit significant quantitative variation for virulence. Despite its importance, the genes responsible for quantitative virulence in this pathogen remain largely unknown. We investigated the expression profiles of four Z. tritici strains differing in virulence in an experiment conducted under uniform environmental conditions. Transcriptomes were compared at four different infection stages to characterize the regulation of gene families thought to be involved in virulence and to identify new virulence factors. The major components of the fungal infection transcriptome showed consistent expression profiles across strains. However, strain-specific regulation was observed for many genes, including some encoding putative virulence factors. We postulate that strain-specific regulation of virulence factors can determine the outcome of Z. tritici infections. We show that differences in gene expression may be major determinants of virulence variation among Z. tritici strains, adding to the already known contributions to virulence variation based on differences in gene sequence and gene presence/absence polymorphisms.

Zymoseptoria tritici causes Septoria tritici blotch (STB) on wheat. The disease interaction is characterized by clearly defined temporal phases of infection, ultimately resulting in the death of host tissue. Zymoseptoria tritici is a highly polymorphic species with significant intraspecific variation in virulence profiles. We generated a deep transcriptomic sequencing dataset spanning the entire time course of an infection using a previously uncharacterized, highly virulent Z. tritici strain isolated from a Swiss wheat field. We found that seven clusters of gene transcription profiles explained the progression of the infection. The earliest highly up-regulated genes included chloroperoxidases, which may help the fungus cope with plant defences. The onset of necrotrophy was characterized by a concerted up-regulation of proteases, plant cell wall-degrading enzymes and lipases. Functions related to nutrition and growth characterized late necrotrophy and the transition to saprotrophic growth on dead plant tissue. We found that the peak up-regulation of genes essential for mating coincided with the necrotrophic phase. We performed an intraspecies comparative transcriptomics analysis using a comparable time course infection experiment of the genome reference isolate IPO323. Major components of the fungal infection transcriptome were conserved between the two strains. However, individual small, secreted proteins, proteases and cell wall-degrading enzymes showed strongly differentiated transcriptional profiles between isolates. Our analyses illustrate that successful STB infections involve complex transcriptomic remodelling to up-regulate distinct gene functions. Heterogeneity in transcriptomes among isolates may explain some of the considerable variation in virulence and host specialization found within the species., (© 2015 BSPP AND JOHN WILEY & SONS LTD.)

Chromosomal rearrangements are a major driver of eukaryotic genome evolution, affecting speciation, pathogenicity and cancer progression. Changes in chromosome structure are often initiated by mis-repair of double-strand breaks in the DNA. Mis-repair is particularly likely when telomeres are lost or when dispersed repeats misalign during crossing-over. Fungi carry highly polymorphic chromosomal complements showing substantial variation in chromosome length and number. The mechanisms driving chromosome polymorphism in fungi are poorly understood. We aimed to identify mechanisms of chromosomal rearrangements in the fungal wheat pathogen Zymoseptoria tritici. We combined population genomic resequencing and chromosomal segment PCR assays with electrophoretic karyotyping and resequencing of parents and offspring from experimental crosses to show that this pathogen harbors a highly diverse complement of accessory chromosomes that exhibits strong global geographic differentiation in numbers and lengths of chromosomes. Homologous chromosomes carried highly differentiated gene contents due to numerous insertions and deletions. The largest accessory chromosome recently doubled in length through insertions totaling 380 kb. Based on comparative genomics, we identified the precise breakpoint locations of these insertions. Nondisjunction during meiosis led to chromosome losses in progeny of three different crosses. We showed that a new accessory chromosome emerged in two viable offspring through a fusion between sister chromatids. Such chromosome fusion is likely to initiate a breakage-fusion-bridge (BFB) cycle that can rapidly degenerate chromosomal structure. We suggest that the accessory chromosomes of Z. tritici originated mainly from ancient core chromosomes through a degeneration process that included BFB cycles, nondisjunction and mutational decay of duplicated sequences. The rapidly evolving accessory chromosome complement may serve as a cradle for adaptive evolution in this and other fungal pathogens., Competing Interests: The authors have declared that no competing interests exist.

Zymoseptoria is a newly described genus that includes the prominent wheat pathogen Zymoseptoria tritici (synonyms Mycosphaerella graminicola and Septoria tritici). Studies indicated that the center of origin of Z. tritici is in the Middle East where this important pathogen emerged during the domestication of wheat. Several Zymoseptoria species have been found on uncultivated grasses in the Middle East, and in this article we describe two new Zymoseptoria species from Iran. These species, isolated from Elymus repens, Dactylis glomerata and Lolium perenne, are named Z. ardabiliae and Z. pseudotritici. Both species were identified by means of morphological characteristics and phylogenetic analyses of a seven-gene DNA dataset. These taxa comprise some of the closest known relatives of the wheat pathogen Z. tritici, confirming the reported close phylogenetic relationship between Z. tritici and Z. pseudotritici.

The fungus Mycosphaerella graminicola emerged as a new pathogen of cultivated wheat during its domestication ~11,000 yr ago. We assembled 12 high-quality full genome sequences to investigate the genetic footprints of selection in this wheat pathogen and closely related sister species that infect wild grasses. We demonstrate a strong effect of natural selection in shaping the pathogen genomes with only ~3% of nonsynonymous mutations being effectively neutral. Forty percent of all fixed nonsynonymous substitutions, on the other hand, are driven by positive selection. Adaptive evolution has affected M. graminicola to the highest extent, consistent with recent host specialization. Positive selection has prominently altered genes encoding secreted proteins and putative pathogen effectors supporting the premise that molecular host-pathogen interaction is a strong driver of pathogen evolution. Recent divergence between pathogen sister species is attested by the high degree of incomplete lineage sorting (ILS) in their genomes. We exploit ILS to generate a genetic map of the species without any crossing data, document recent times of species divergence relative to genome divergence, and show that gene-rich regions or regions with low recombination experience stronger effects of natural selection on neutral diversity. Emergence of a new agricultural host selected a highly specialized and fast-evolving pathogen with unique evolutionary patterns compared with its wild relatives. The strong impact of natural selection, we document, is at odds with the small effective population sizes estimated and suggest that population sizes were historically large but likely unstable.

The fungus Mycosphaerella graminicola has been a pathogen of wheat since host domestication 10,000-12,000 years ago in the Fertile Crescent. The wheat-infecting lineage emerged from closely related Mycosphaerella pathogens infecting wild grasses. We use a comparative genomics approach to assess how the process of host specialization affected the genome structure of M. graminicola since divergence from the closest known progenitor species named M. graminicola S1. The genome of S1 was obtained by Illumina sequencing resulting in a 35 Mb draft genome sequence of 32X. Assembled contigs were aligned to the previously sequenced M. graminicola genome. The alignment covered >90% of the non-repetitive portion of the M. graminicola genome with an average divergence of 7%. The sequenced M. graminicola strain is known to harbor thirteen essential chromosomes plus eight dispensable chromosomes. We found evidence that structural rearrangements significantly affected the dispensable chromosomes while the essential chromosomes were syntenic. At the nucleotide level, the essential and dispensable chromosomes have evolved differently. The average synonymous substitution rate in dispensable chromosomes is considerably lower than in essential chromosomes, whereas the average non-synonymous substitution rate is three times higher. Differences in molecular evolution can be related to different transmission and recombination patterns, as well as to differences in effective population sizes of essential and dispensable chromosomes. In order to identify genes potentially involved in host specialization or speciation, we calculated ratios of synonymous and non-synonymous substitution rates in the >9,500 aligned protein coding genes. The genes are generally under strong purifying selection. We identified 43 candidate genes showing evidence of positive selection, one encoding a potential pathogen effector protein. We conclude that divergence of these pathogens was accompanied by structural rearrangements in the small dispensable chromosomes, while footprints of positive selection were present in only a small number of protein coding genes., Competing Interests: The authors have declared that no competing interests exist.

Rhynchosporium secalis is an important pathogen of barley globally. Fourteen polymorphic microsatellites were analyzed for 1664 R. secalis isolates sampled from 37 field populations to infer their demographic history. The results falsified the hypothesis that R. secalis co-evolved with its barley host in the Middle East. Populations from Scandinavia had significantly higher allelic diversities, the greatest number of private alleles and the highest genotypic diversities. All but three of the analyzed populations had an excess of gene diversity compared to the number of alleles, consistent with a recent population bottleneck. The remaining populations had a gene diversity deficit consistent with a population expansion following a recent population bottleneck in the last +/-100 years. A coalescent analysis revealed that the effective population sizes based on theta, of the analyzed populations were small relative to their ancestral population sizes, indicating that only a fraction of the diversity present in the ancestral populations was transmitted into current populations. These findings are consistent with the hypothesis that the pathogen population on barley experienced a selection bottleneck imposed by the host and/or are founder populations. The mean estimate of migration rates was 2.2 (avg 90% confidence interval=1.3-3.1). Major migration routes were identified among populations separated by long distances, eg between South Africa and Australia, as well as among North Africa, the Middle East and California, suggesting contemporary exchange of infected barley seed. In contrast with earlier findings, most populations exhibited significant gametic disequilibrium, probably as a result of genetic drift. We conclude that the majority of R. secalis populations have experienced human-mediated migration that led to numerous and relatively recent founder events around the world.

Aims: To develop a molecular identification method based on ISSR fingerprints to monitor the fungal leaf pathogen Stagonospora convolvuli LA39 used to biologically control bindweeds after a field release., Methods and Results: The developed method proved to be suitable to clearly distinguish LA39 from resident Stagonospora spp. and was applied in two field experiments. First, the environmental persistence of LA39 was assessed in an overwintering experiment. LA39 could be re-isolated from infected bindweed 1 year after field application, but with very low frequency of occurrence. Secondly, LA39 was applied in an area with natural bindweed infestation and re-isolated from infected bindweed. The dispersal of LA39 during one season was poor (4-5 m)., Conclusions: ISSR fingerprinting has been shown to be a valuable tool to monitor the environmental fate of S. convolvuli in the field. It is concluded that an LA39-based mycoherbicide will have minimal environmental impact caused by the restricted mobility, poor proliferation and poor persistence over seasons of LA39., Significance and Impact of the Study: Studies about the dispersal and survival of biocontrol agents after field release as well as the development of methods needed for this purpose are indispensable for a comprehensive risk assessment for biocontrol agents.

Rhynchosporium secalis, the causal agent of scald on barley, is thought to be exclusively asexual because no teleomorph has been found. Partial sequences of the HMG-box and alpha-domain of Rhynchosporium secalis isolates were identified and used to develop a PCR assay for the mating-type locus. PCR amplification of only one of these two domains was possible in each strain, suggesting that R. secalis has a MAT organization that is similar to other known heterothallic fungi. A multiplex PCR with primers amplifying either a MAT1-1- or MAT1-2-specific amplicon was used to determine the distribution of mating types in several R. secalis populations. In total, 1101 isolates from Australia, Switzerland, Ethiopia, Scandinavia, California, and South Africa were included in the analysis. Mating types occurred in equal frequencies for most of these populations, suggesting frequency-dependent selection consistent with sexual reproduction. In addition, both mating types were frequently found occupying the same lesion or leaf, providing opportunities for isolates of opposite mating type to interact and reproduce sexually. We propose that R. secalis should be considered a sexual pathogen, although the sexual cycle may occur infrequently in some populations.