Repeat-induced Point Mutation Research Articles

Repeat-induced point mutations driving Parastagonospora nodorum genomic diversity are balanced by selection against non-synonymous mutations

Parastagonospora nodorum is necrotrophic fungal pathogen of wheat with significant genomic resources. Population-level pangenome data for 173 isolates, of which 156 were from Western Australia (WA) and 17 were international, were examined for overall genomic diversity and effector gene content. A heterothallic core population occurred across all regions of WA, with asexually-reproducing clonal clusters in dryer northern regions. High potential for SNP diversity in the form of repeat-induced point mutation (RIP)-like transitions, was observed across the genome, suggesting widespread ‘RIP-leakage’ from transposon-rich repetitive sequences into non-repetitive regions. The strong potential for RIP-like mutations was balanced by negative selection against non-synonymous SNPs, that was observed within protein-coding regions. Protein isoform profiles of known effector loci (SnToxA, SnTox1, SnTox3, SnTox267, and SnTox5) indicated low-levels of non-synonymous and high-levels of silent RIP-like mutations. Effector predictions identified 186 candidate secreted predicted effector proteins (CSEPs), 69 of which had functional annotations and included confirmed effectors. Pangenome-based effector isoform profiles across WA were distinct from global isolates and were conserved relative to population structure, and may enable new approaches for monitoring crop disease pathotypes.

Translation of Mutant Repetitive Genomic Sequences in Hirsutella sinensis and Changes in the Secondary Structures and Functional Specifications of the Encoded Proteins.

Multiple repetitive sequences of authentic genes commonly exist in fungal genomes. AT-biased genotypes of Ophiocordyceps sinensis have been hypothesized as repetitive pseudogenes in the genome of Hirsutella sinensis (GC-biased Genotype #1 of O. sinensis) and are generated through repeat-induced point mutation (RIP), which is charactered by cytosine-to-thymine and guanine-to-adenine transitions, concurrent epigenetic methylation, and dysfunctionality. This multilocus study examined repetitive sequences in the H. sinensis genome and transcriptome using a bioinformatic approach and revealed that 8.2% of the authentic genes had repetitive copies, including various allelic insertions/deletions, transversions, and transitions. The transcripts for the repetitive sequences, regardless of the decreases, increases, or bidirectional changes in the AT content, were identified in the H. sinensis transcriptome, resulting in changes in the secondary protein structure and functional specification. Multiple repetitive internal transcribed spacer (ITS) copies containing multiple insertion/deletion and transversion alleles in the genome of H. sinensis were GC-biased and were theoretically not generated through RIP mutagenesis. The repetitive ITS copies were genetically and phylogenetically distinct from the AT-biased O. sinensis genotypes that possess multiple transition alleles. The sequences of Genotypes #2-17 of O. sinensis, both GC- and AT-biased, were absent from the H. sinensis genome, belong to the interindividual fungi, and differentially occur in different compartments of the natural Cordyceps sinensis insect-fungi complex, which contains >90 fungal species from >37 genera. Metatranscriptomic analyses of natural C. sinensis revealed the transcriptional silencing of 5.8S genes in all C. sinensis-colonizing fungi in natural settings, including H. sinensis and other genotypes of O. sinensis. Thus, AT-biased genotypes of O. sinensis might have evolved through advanced evolutionary mechanisms, not through RIP mutagenesis, in parallel with GC-biased Genotype #1 of H. sinensis from a common genetic ancestor over the long course of evolution.

Genome-wide survey of the bipartite structure and pathogenesis-related genes of Neostagonosporella sichuanensis, a causal agent of Fishscale bamboo rhombic-spot disease.

Bamboo resources have garnered significant global attention due to their excellent capacity for regeneration and high yield. Rhombic-spot disease, a substantial threat to fishscale bamboo (Phyllostachys heteroclada), is primarily caused by Neostagonosporella sichuanensis. This study first reported the genome assemblies and characteristics of two N. sichuanensis isolates using PacBio and Illumina sequencing platforms. The genomes of N. sichuanensis strain SICAUCC 16-0001 and strain SICAUCC 23-0140, with sizes of 48.0 Mb and 48.4 Mb, respectively, revealed 10,289 and 10,313 protein-coding genes. Additionally, they contained 34.99 and 34.46% repetitive sequences within AT-rich regions, with notable repeat-induced point mutation activity. Comparative genome analysis identified 1,049 contracted and 45 expanded gene families in the genome of N. sichuanensis, including several related to pathogenicity. Several gene families involved in mycotoxin metabolism, secondary metabolism, sterol biosynthesis and transport, and cell wall degradation were contracted. Compared to most analyzed necrotrophic, hemibiotrophic, and phaeosphaeriacous pathogens, the genomes of two N. sichuanensis isolates exhibited fewer secondary metabolite enzymes, carbohydrate-active enzymes, plant cell wall degrading enzymes, secreted proteins, and effectors. Comparative genomics analysis suggested that N. sichuanensis shares more similar characteristics with hemibiotrophic pathogens. Based on single carbon source tests, N. sichuanensis strains demonstrated a higher potential for xylan decomposition than pectin and cellulose. The proportion of cell wall-degrading enzyme effectors occupied a high proportion of the total effectors of the N. sichuanensis genomes. These findings provide valuable insights into uncovering the pathogenesis of N. sichuanensis toward the efficient management of rhombic-spot disease of fishscale bamboo.

Highly Repetitive Genome of Coniella granati (syn. Pilidiella granati), the Causal Agent of Pomegranate Fruit Rot, Encodes a Minimalistic Proteome with a Streamlined Arsenal of Effector Proteins.

This study describes the first genome sequence and analysis of Coniella granati, a fungal pathogen with a broad host range, which is responsible for postharvest crown rot, shoot blight, and canker diseases in pomegranates. C. granati is a geographically widespread pathogen which has been reported across Europe, Asia, the Americas, and Africa. Our analysis revealed a 46.8 Mb genome with features characteristic of hemibiotrophic fungi. Approximately one third of its genome was compartmentalised within 'AT-rich' regions exhibiting a low GC content (30 to 45%). These regions primarily comprised transposable elements that are repeated at a high frequency and interspersed throughout the genome. Transcriptome-supported gene annotation of the C. granati genome revealed a streamlined proteome, mirroring similar observations in other pathogens with a latent phase. The genome encoded a relatively compact set of 9568 protein-coding genes with a remarkable 95% having assigned functional annotations. Despite this streamlined nature, a set of 40 cysteine-rich candidate secreted effector-like proteins (CSEPs) was predicted as well as a gene cluster involved in the synthesis of a pomegranate-associated toxin. These potential virulence factors were predominantly located near repeat-rich and AT-rich regions, suggesting that the pathogen evades host defences through Repeat-Induced Point mutation (RIP)-mediated pseudogenisation. Furthermore, 23 of these CSEPs exhibited homology to known effector and pathogenicity genes found in other hemibiotrophic pathogens. The study establishes a foundational resource for the study of the genetic makeup of C. granati, paving the way for future research on its pathogenicity mechanisms and the development of targeted control strategies to safeguard pomegranate production.

H3T11 phosphorylation by CKII is required for heterochromatin formation in Neurospora.

Heterochromatin is a key feature of eukaryotic genomes and is crucial for maintaining genomic stability. In fission yeast, heterochromatin nucleation is mainly mediated by DNA-binding proteins or the RNA interference (RNAi) pathway. In the filamentous fungus Neurospora crassa, however, the mechanism that causes the initiation of heterochromatin at the relics of repeat-induced point mutation is unknown and independent of the classical RNAi pathway. Here, we show that casein kinase II (CKII) and its kinase activity are required for heterochromatin formation at the well-defined 5-kb heterochromatin of the 5H-cat-3 region and transcriptional repression of its adjacent cat-3 gene. Similarly, mutation of the histone H3 phosphorylation site T11 also impairs heterochromatin formation at the same locus. The catalytic subunit CKA colocalizes with H3T11phosphorylation (H3pT11) within the 5H-cat-3 domain and the deletion of cka results in a significant decrease in H3T11 phosphorylation. Furthermore, the loss of kinase activity of CKII results in a significant reduction of H3pT11, H3K9me3 (histone H3 lysine 9 trimethylation)and DNA methylation levels, suggesting that CKII regulates heterochromatin formation by promoting H3T11 phosphorylation. Together, our results establish that histone H3 phosphorylation by CKII is a critical event required for heterochromatin formation.

Update on the Basic Understanding of Fusarium graminearum Virulence Factors in Common Wheat Research.

Wheat is one of the most important food crops, both in China and worldwide. Wheat production is facing extreme stresses posed by different diseases, including Fusarium head blight (FHB), which has recently become an increasingly serious concerns. FHB is one of the most significant and destructive diseases affecting wheat crops all over the world. Recent advancements in genomic tools provide a new avenue for the study of virulence factors in relation to the host plants. The current review focuses on recent progress in the study of different strains of Fusarium infection. The presence of genome-wide repeat-induced point (RIP) mutations causes genomic mutations, eventually leading to host plant susceptibility against Fusarium invasion. Furthermore, effector proteins disrupt the host plant resistance mechanism. In this study, we proposed systematic modification of the host genome using modern biological tools to facilitate plant resistance against foreign invasion. We also suggested a number of scientific strategies, such as gene cloning, developing more powerful functional markers, and using haplotype marker-assisted selection, to further improve FHB resistance and associated breeding methods.

The massive 340 megabase genome of Anisogramma anomala, a biotrophic ascomycete that causes eastern filbert blight of hazelnut

BackgroundThe ascomycete fungus Anisogramma anomala causes Eastern Filbert Blight (EFB) on hazelnut (Corylus spp.) trees. It is a minor disease on its native host, the American hazelnut (C. americana), but is highly destructive on the commercially important European hazelnut (C. avellana). In North America, EFB has historically limited commercial production of hazelnut to west of the Rocky Mountains. A. anomala is an obligately biotrophic fungus that has not been grown in continuous culture, rendering its study challenging. There is a 15-month latency before symptoms appear on infected hazelnut trees, and only a sexual reproductive stage has been observed. Here we report the sequencing, annotation, and characterization of its genome.ResultsThe genome of A. anomala was assembled into 108 scaffolds totaling 342,498,352 nt with a GC content of 34.46%. Scaffold N50 was 33.3 Mb and L50 was 5. Nineteen scaffolds with lengths over 1 Mb constituted 99% of the assembly. Telomere sequences were identified on both ends of two scaffolds and on one end of another 10 scaffolds. Flow cytometry estimated the genome size of A. anomala at 370 Mb. The genome exhibits two-speed evolution, with 93% of the assembly as AT-rich regions (32.9% GC) and the other 7% as GC-rich (57.1% GC). The AT-rich regions consist predominantly of repeats with low gene content, while 90% of predicted protein coding genes were identified in GC-rich regions. Copia-like retrotransposons accounted for more than half of the genome. Evidence of repeat-induced point mutation (RIP) was identified throughout the AT-rich regions, and two copies of the rid gene and one of dim-2, the key genes in the RIP mutation pathway, were identified in the genome. Consistent with its homothallic sexual reproduction cycle, both MAT1-1 and MAT1-2 idiomorphs were found. We identified a large suite of genes likely involved in pathogenicity, including 614 carbohydrate active enzymes, 762 secreted proteins and 165 effectors.ConclusionsThis study reveals the genomic structure, composition, and putative gene function of the important pathogen A. anomala. It provides insight into the molecular basis of the pathogen’s life cycle and a solid foundation for studying EFB.

No safe haven: Loss of avirulence in the plant pathogen Leptosphaeria maculans by DNA duplication and repeat‐induced point mutation

AbstractMicrobes can overcome the ability of plant resistance genes to confer protection against disease by mutating their corresponding avirulence genes. The fungus Leptosphaeria maculans causes blackleg disease on canola crops and subverts Brassica napus resistance genes through several DNA mutation mechanisms. One of these is repeat‐induced point (RIP) mutation, which can ‘leak’ into the avirulence genes from the adjacent repetitive sequences that the mutation process is targeting. Here, we identified populations of L. maculans in Australia that have extensive RIP mutations in the avirulence gene AvrLm2 and show that this has been triggered by a duplication of the gene and surrounding DNA that includes the distant (>55 kb in total) AvrLm6 gene. This finding provides another mechanism of mutation by which pathogens can overcome host resistance, and more broadly contributes to understanding the complex balance between gene duplication and genome defence.

Recombination and repeat-induced point mutation landscapes reveal trade-offs between the sexual and asexual cycles of Magnaporthe oryzae

The fungal disease caused by Magnaporthe oryzae is one of the most devastating diseases that endanger many crops worldwide. Evidence shows that sexual reproduction can be advantageous for fungal diseases as hybridization facilitates host-jumping. However, the pervasive clonal lineages of M. oryzae observed in natural fields contradict this expectation. A better understanding of the roles of recombination and the fungi-specific repeat-induced point mutation (RIP) in shaping its evolutionary trajectory is essential to bridge this knowledge gap. Here we systematically investigate the RIP and recombination landscapes in M. oryzae using a whole genome sequencing data from 252 population samples and 92 cross progenies. Our data reveal that the RIP can robustly capture the population history of M. oryzae, and we provide accurate estimations of the recombination and RIP rates across different M. oryzae clades. Significantly, our results highlight a parent-of-origin bias in both recombination and RIP rates, tightly associating with their sexual potential and variations of effector proteins. This bias suggests a critical trade-off between generating novel allelic combinations in the sexual cycle to facilitate host-jumping and stimulating transposon-associated diversification of effectors in the asexual cycle to facilitate host coevolution. These findings provide unique insights into understanding the evolution of blast fungus.

Sexual stage–specific A-to-I mRNA editing is mediated by tRNA-editing enzymes in fungi

A-to-I RNA editing catalyzed by adenosine-deaminase-acting-on-RNA (ADARs) was assumed to be unique to metazoans because fungi and plants lack ADAR homologs. However, genome-wide messenger RNA (mRNA) editing was found to occur specifically during sexual reproduction in filamentous ascomycetes. Because systematic characterization of adenosine/cytosine deaminase genes has implicated the involvement of TAD2 and TAD3 orthologs in A-to-I editing, in this study, we used genetic and biochemical approaches to characterize the role of FgTAD2, an essential adenosine-deaminase-acting-on-tRNA (ADAT) gene, in mRNA editing in Fusarium graminearum. FgTAD2 had a sexual-stage-specific isoform and formed heterodimers with enzymatically inactive FgTAD3. Using a repeat-induced point (RIP) mutation approach, we identified 17 mutations in FgTAD2 that affected mRNA editing during sexual reproduction but had no effect on transfer RNA (tRNA) editing and vegetative growth. The functional importance of the H352Y and Q375*(nonsense) mutations in sexual reproduction and mRNA editing were confirmed by introducing specific point mutations into the endogenous FgTAD2 allele in the wild type. An in vitro assay was developed to show that FgTad2-His proteins purified from perithecia, but not from vegetative hyphae, had mRNA editing activities. Moreover, the H352Y mutation affected the enzymatic activity of FgTad2 to edit mRNA but had no effect on its ADAT activity. We also identified proteins co-purified with FgTad2-His by mass spectrometry analysis and found that two of them have the RNA recognition motif. Taken together, genetic and biochemical data from this study demonstrated that FgTad2, an ADAT, catalyzes A-to-I mRNA editing with the stage-specific isoform and cofactors during sexual reproduction in fungi.

Analysis of five near-complete genome assemblies of the tomato pathogen Cladosporium fulvum uncovers additional accessory chromosomes and structural variations induced by transposable elements effecting the loss of avirulence genes

BackgroundFungal plant pathogens have dynamic genomes that allow them to rapidly adapt to adverse conditions and overcome host resistance. One way by which this dynamic genome plasticity is expressed is through effector gene loss, which enables plant pathogens to overcome recognition by cognate resistance genes in the host. However, the exact nature of these loses remains elusive in many fungi. This includes the tomato pathogen Cladosporium fulvum, which is the first fungal plant pathogen from which avirulence (Avr) genes were ever cloned and in which loss of Avr genes is often reported as a means of overcoming recognition by cognate tomato Cf resistance genes. A recent near-complete reference genome assembly of C. fulvum isolate Race 5 revealed a compartmentalized genome architecture and the presence of an accessory chromosome, thereby creating a basis for studying genome plasticity in fungal plant pathogens and its impact on avirulence genes.ResultsHere, we obtained near-complete genome assemblies of four additional C. fulvum isolates. The genome assemblies had similar sizes (66.96 to 67.78 Mb), number of predicted genes (14,895 to 14,981), and estimated completeness (98.8 to 98.9%). Comparative analysis that included the genome of isolate Race 5 revealed high levels of synteny and colinearity, which extended to the density and distribution of repetitive elements and of repeat-induced point (RIP) mutations across homologous chromosomes. Nonetheless, structural variations, likely mediated by transposable elements and effecting the deletion of the avirulence genes Avr4E, Avr5, and Avr9, were also identified. The isolates further shared a core set of 13 chromosomes, but two accessory chromosomes were identified as well. Accessory chromosomes were significantly smaller in size, and one carried pseudogenized copies of two effector genes. Whole-genome alignments further revealed genomic islands of near-zero nucleotide diversity interspersed with islands of high nucleotide diversity that co-localized with repeat-rich regions. These regions were likely generated by RIP, which generally asymmetrically affected the genome of C. fulvum.ConclusionsOur results reveal new evolutionary aspects of the C. fulvum genome and provide new insights on the importance of genomic structural variations in overcoming host resistance in fungal plant pathogens.

Evolutionary dynamics of the LTR-retrotransposon crapaud in the Podospora anserina species complex and the interaction with repeat-induced point mutations

BackgroundThe genome of the filamentous ascomycete Podospora anserina shows a relatively high abundance of retrotransposons compared to other interspersed repeats. The LTR-retrotransposon family crapaud is particularly abundant in the genome, and consists of multiple diverged sequence variations specifically localized in the 5’ half of both long terminal repeats (LTRs). P. anserina is part of a recently diverged species-complex, which makes the system ideal to classify the crapaud family based on the observed LTR variation and to study the evolutionary dynamics, such as the diversification and bursts of the elements over recent evolutionary time.ResultsWe developed a sequence similarity network approach to classify the crapaud repeats of seven genomes representing the P. anserina species complex into 14 subfamilies. This method does not utilize a consensus sequence, but instead it connects any copies that share enough sequence similarity over a set sequence coverage. Based on phylogenetic analyses, we found that the crapaud repeats likely diversified in the ancestor of the complex and have had activity at different time points for different subfamilies. Furthermore, while we hypothesized that the evolution into multiple subfamilies could have been a direct effect of escaping the genome defense system of repeat induced point mutations, we found this not to be the case.ConclusionsOur study contributes to the development of methods to classify transposable elements in fungi, and also highlights the intricate patterns of retrotransposon evolution over short timescales and under high mutational load caused by nucleotide-altering genome defense.

The Hidden Truths of Fungal Virulence and Adaptation on Hosts: Unraveling the Conditional Dispensability of Minichromosomes in the Hemibiotrophic Colletotrichum Pathogens.

Colletotrichum spp. are ascomycete fungi and cause anthracnose disease in numerous crops of economic significance. The genomes of these fungi are distributed among ten core chromosomes and two to three minichromosomes. While the core chromosomes regulate fungal growth, development and virulence, the extent to which the minichromosomes are involved in these processes is still uncertain. Here, we discuss the minichromosomes of three hemibiotrophic Colletotrichum pathogens, i.e., C. graminicola, C. higginsianum and C. lentis. These minichromosomes are typically less than one megabase in length, characterized by containing higher repetitive DNA elements, lower GC content, higher frequency of repeat-induced point mutations (RIPMs) and sparse gene distribution. Molecular genetics and functional analyses have revealed that these pathogens harbor one conditionally dispensable minichromosome, which is dispensable for fungal growth and development but indispensable for fungal virulence on hosts. They appear to be strain-specific innovations and are highly compartmentalized into AT-rich and GC-rich blocks, resulting from RIPMs, which may help protect the conditionally dispensable minichromosomes from erosion of already scarce genes, thereby helping the Colletotrichum pathogens maintain adaptability on hosts. Overall, understanding the mechanisms underlying the conditional dispensability of these minichromosomes could lead to new strategies for controlling anthracnose disease in crops.

Recombination hotspots in Neurospora crassa controlled by idiomorphic sequences and meiotic silencing.

Genes regulating recombination in specific chromosomal intervals of Neurospora crassa were described in the 1960s, but the mechanism is still unknown. For each of the rec-1, rec-2, and rec-3 genes, a single copy of the putative dominant allele, for example, rec-2SL found in St Lawrence OR74 A wild type, reduces recombination in chromosomal regions specific to that gene. However, when we sequenced the recessive allele, rec-2LG (derived from the Lindegren 1A wild type), we found that a 10 kb region in rec-2SL strains was replaced by a 2.7 kb unrelated sequence, making the "alleles" idiomorphs. When we introduced sad-1, a mutant lacking the RNA-dependent RNA polymerase that silences unpaired coding regions during meiosis into crosses heterozygous rec-2SL/rec-2LG, it increased recombination, indicating that meiotic silencing of a gene promoting recombination is responsible for dominant suppression of recombination. Consistent with this, mutation of rec-2LG by Repeat-Induced Point mutation generated an allele with multiple stop codons in the predicted rec-2 gene, which does not promote recombination and is recessive to rec-2LG. Sad-1 also relieves suppression of recombination in relevant target regions, in crosses heterozygous for rec-1 alleles and in crosses heterozygous for rec-3 alleles. We conclude that for all 3 known rec genes, 1 allele appears dominant only because meiotic silencing prevents the product of the active, "recessive," allele from stimulating recombination during meiosis. In addition, the proposed amino acid sequence of REC-2 suggests that regulation of recombination in Neurospora differs from any currently known mechanism.

Long noncoding RNAs emerge from transposon-derived antisense sequences and may contribute to infection stage-specific transposon regulation in a fungal phytopathogen

BackgroundThe genome of the obligate biotrophic phytopathogenic barley powdery mildew fungus Blumeria hordei is inflated due to highly abundant and possibly active transposable elements (TEs). In the absence of the otherwise common repeat-induced point mutation transposon defense mechanism, noncoding RNAs could be key for regulating the activity of TEs and coding genes during the pathogenic life cycle.ResultsWe performed time-course whole-transcriptome shotgun sequencing (RNA-seq) of total RNA derived from infected barley leaf epidermis at various stages of fungal pathogenesis and observed significant transcript accumulation and time point-dependent regulation of TEs in B. hordei. Using a manually curated consensus database of 344 TEs, we discovered phased small RNAs mapping to 104 consensus transposons, suggesting that RNA interference contributes significantly to their regulation. Further, we identified 5,127 long noncoding RNAs (lncRNAs) genome-wide in B. hordei, of which 823 originated from the antisense strand of a TE. Co-expression network analysis of lncRNAs, TEs, and coding genes throughout the asexual life cycle of B. hordei points at extensive positive and negative co-regulation of lncRNAs, subsets of TEs and coding genes.ConclusionsOur work suggests that similar to mammals and plants, fungal lncRNAs support the dynamic modulation of transcript levels, including TEs, during pivotal stages of host infection. The lncRNAs may support transcriptional diversity and plasticity amid loss of coding genes in powdery mildew fungi and may give rise to novel regulatory elements and virulence peptides, thus representing key drivers of rapid evolutionary adaptation to promote pathogenicity and overcome host defense.

Genome-scale phylogeny and comparative genomics of the fungal order Sordariales

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking.In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae.Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.

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, similar to the effect of the chromatin conformation on the mitotic mutation rate. To date, however, the exact distribution and type of meiosis-associated changes and the role of the chromatin conformation in this context are 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 confirm a high genome-wide recombination rate of 65 centimorgan (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 approximately 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. IMPORTANCE The 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.

Starships are active eukaryotic transposable elements mobilized by a new family of tyrosine recombinases

Transposable elements in eukaryotic organisms have historically been considered "selfish," at best conferring indirect benefits to their host organisms. The Starships are a recently discovered feature in fungal genomes that are, in some cases, predicted to confer beneficial traits to their hosts and also have hallmarks of being transposable elements. Here, we provide experimental evidence that Starships are indeed autonomous transposons, using the model Paecilomyces variotii, and identify the HhpA "Captain" tyrosine recombinase as essential for their mobilization into genomic sites with a specific target site consensus sequence. Furthermore, we identify multiple recent horizontal gene transfers of Starships, implying that they jump between species. Fungal genomes have mechanisms to defend against mobile elements, which are frequently detrimental to the host. We discover that Starships are also vulnerable to repeat-induced point mutation defense, thereby having implications on the evolutionary stability of such elements.

Chromosome-level analysis of the Colletotrichum graminicola genome reveals the unique characteristics of core and minichromosomes.

The fungal pathogen Colletotrichum graminicola causes the anthracnose of maize (Zea mays) and is responsible for significant yield losses worldwide. The genome of C. graminicola was sequenced in 2012 using Sanger sequencing, 454 pyrosequencing, and an optical map to obtain an assembly of 13 pseudochromosomes. We re-sequenced the genome using a combination of short-read (Illumina) and long-read (PacBio) technologies to obtain a chromosome-level assembly. The new version of the genome sequence has 13 chromosomes with a total length of 57.43 Mb. We detected 66 (23.62 Mb) structural rearrangements in the new assembly with respect to the previous version, consisting of 61 (21.98 Mb) translocations, 1 (1.41 Mb) inversion, and 4 (221 Kb) duplications. We annotated the genome and obtained 15,118 predicted genes and 3,614 new gene models compared to the previous version of the assembly. We show that 25.88% of the new assembly is composed of repetitive DNA elements (13.68% more than the previous assembly version), which are mostly found in gene-sparse regions. We describe genomic compartmentalization consisting of repeat-rich and gene-poor regions vs. repeat-poor and gene-rich regions. A total of 1,140 secreted proteins were found mainly in repeat-rich regions. We also found that ~75% of the three smallest chromosomes (minichromosomes, between 730 and 551 Kb) are strongly affected by repeat-induced point mutation (RIP) compared with 28% of the larger chromosomes. The gene content of the minichromosomes (MCs) comprises 121 genes, of which 83.6% are hypothetical proteins with no predicted function, while the mean percentage of Chr1-Chr10 is 36.5%. No predicted secreted proteins are present in the MCs. Interestingly, only 2% of the genes in Chr11 have homologs in other strains of C. graminicola, while Chr12 and 13 have 58 and 57%, respectively, raising the question as to whether Chrs12 and 13 are dispensable. The core chromosomes (Chr1-Chr10) are very different with respect to the MCs (Chr11-Chr13) in terms of the content and sequence features. We hypothesize that the higher density of repetitive elements and RIPs in the MCs may be linked to the adaptation and/or host co-evolution of this pathogenic fungus.

Investigating the origin of subtelomeric and centromeric AT-rich elements in Aspergillus flavus.

An in silico study of Aspergillus flavus genome stability uncovered significant variations in both coding and non-coding regions. The non-coding insertions uniformly consisted of AT-rich sequences that are evolutionarily maintained, albeit distributed at widely different sites in an array of A. flavus strains. A survey of ≥ 2kb AT-rich elements (AT ≥ 70%; ATEs) in non-centromeric regions uncovered two major categories of ATEs. The first category is composed of homologous insertions at ectopic, non-allelic sites that contain homology to transposable elements (TEs; Classes B, C, D, and E). Strains differed significantly in frequency, position, and TE type, but displayed a common enrichment in subtelomeric regions. The TEs were heavily mutated, with patterns consistent with the ancestral activity of repeat-induced point mutations (RIP). The second category consists of a conserved set of novel subtelomeric ATE repeats (Classes A, G, G, H, I and J) which lack discernible TEs and, unlike TEs, display a constant polarity relative to the telomere. Members of one of these classes are derivatives of a progenitor ATE that is predicted to have undergone extensive homologous recombination during evolution. A third category of ATEs consists of ~100 kb regions at each centromere. Centromeric ATEs and TE clusters within these centromeres display a high level of sequence identity between strains. These studies suggest that transposition and RIP are forces in the evolution of subtelomeric and centromeric structure and function.