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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, Metarhizium 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 accessory chromosome that was transferred contains genes that may be involved in its preferential horizontal transfer or support its establishment. These genes encode putative histones and histone-modifying enzymes, as well as putative virulence factors. 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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., Competing Interests: The authors declare no conflict of interest.

Non-Mendelian transmission has been reported for various genetic elements, ranging from small transposons to entire chromosomes. One prime example of such a transmission pattern are B chromosomes in plants and animals. Accessory chromosomes in fungi are similar to B chromosomes in showing presence/absence polymorphism and being non-essential. How these chromosomes are transmitted during meiosis is however poorly understood-despite their often high impact on the fitness of the host. For several fungal organisms, a non-Mendelian transmission or a mechanistically unique meiotic drive of accessory chromosomes have been reported. In this review, we provide an overview of the possible mechanisms that can cause the non-Mendelian transmission or meiotic drives of fungal accessory chromosomes. We compare processes responsible for the non-Mendelian transmission of accessory chromosomes for different fungal eukaryotes and discuss the structural traits of fungal accessory chromosomes affecting their meiotic transmission. We conclude that research on fungal accessory chromosomes, due to their small size, ease of sequencing, and epigenetic profiling, can complement the study of B chromosomes in deciphering factors that influence and regulate the non-Mendelian transmission of entire chromosomes., (© 2022. The Author(s).)

Mutations are the source of genetic variation and the substrate for evolution. Genome-wide mutation rates appear to be affected by selection and are probably adaptive. Mutation rates are also known to vary along genomes, possibly in response to epigenetic modifications, but causality is only assumed. In this study we determine the direct impact of epigenetic modifications and temperature stress on mitotic mutation rates in a fungal pathogen using a mutation accumulation approach. Deletion mutants lacking epigenetic modifications confirm that histone mark H3K27me3 increases whereas H3K9me3 decreases the mutation rate. Furthermore, cytosine methylation in transposable elements (TE) increases the mutation rate 15-fold resulting in significantly less TE mobilization. Also accessory chromosomes have significantly higher mutation rates. Finally, we find that temperature stress substantially elevates the mutation rate. Taken together, we find that epigenetic modifications and environmental conditions modify the rate and the location of spontaneous mutations in the genome and alter its evolutionary trajectory., (© 2021. The Author(s).)

In host-pathogen interactions RNA interference (RNAi) has emerged as a pivotal mechanism to modify both, the immune responses of the host as well as the pathogenicity and virulence of the pathogen. In addition, in some fungi RNAi is also known to affect chromosome biology via its effect on chromatin conformation. Previous studies reported no effect of the RNAi machinery on the virulence of the fungal plant pathogen Zymoseptoria tritici however the role of RNAi is still poorly understood in this species. Herein, we elucidate whether the RNAi machinery is conserved within the genus Zymoseptoria . Moreover, we conduct functional analyses of Argonaute and Dicer-like proteins and test if the RNAi machinery affects chromosome stability. We show that the RNAi machinery is conserved among closely related Zymoseptoria species while an exceptional pattern of allelic diversity was possibly caused by introgression. The deletion of Ago1 reduced the ability of the fungus to produce asexual propagules in planta in a quantitative matter. Chromosome stability of the accessory chromosome of Z. tritici was not prominently affected by the RNAi machinery. These results indicate, in contrast to previous finding, a role of the RNAi pathway during host infection, but not in the stability of accessory chromosomes in Z. tritici .

DNA methylation is found throughout all domains of life, yet the extent and function of DNA methylation differ among eukaryotes. Strains of the plant pathogenic fungus Zymoseptoria tritici appeared to lack cytosine DNA methylation (5mC) because gene amplification followed by Repeat-Induced Point mutation (RIP) resulted in the inactivation of the dim2 DNA methyltransferase gene. 5mC is, however, present in closely related sister species. We demonstrate that inactivation of dim2 occurred recently as some Z. tritici isolates carry a functional dim2 gene. Moreover, we show that dim2 inactivation occurred by a different path than previously hypothesized. We mapped the genome-wide distribution of 5mC in strains with or without functional dim2 alleles. Presence of functional dim2 correlates with high levels of 5mC in transposable elements (TEs), suggesting a role in genome defense. We identified low levels of 5mC in strains carrying non-functional dim2 alleles, suggesting that 5mC is maintained over time, presumably by an active Dnmt5 DNA methyltransferase. Integration of a functional dim2 allele in strains with mutated dim2 restored normal 5mC levels, demonstrating de novo cytosine methylation activity of Dim2. To assess the importance of 5mC for genome evolution, we performed an evolution experiment, comparing genomes of strains with high levels of 5mC to genomes of strains lacking functional dim2. We found that presence of a functional dim2 allele alters nucleotide composition by promoting C to T transitions (C→T) specifically at CpA (CA) sites during mitosis, likely contributing to TE inactivation. Our results show that 5mC density at TEs is a polymorphic trait in Z. tritici populations that can impact genome evolution., Competing Interests: The authors have declared that no competing interests exist.

Background: Antagonistic co-evolution can drive rapid adaptation in pathogens and shape genome architecture. Comparative genome analyses of several fungal pathogens revealed highly variable genomes, for many species characterized by specific repeat-rich genome compartments with exceptionally high sequence variability. Dynamic genome structure may enable fast adaptation to host genetics. The wheat pathogen Zymoseptoria tritici with its highly variable genome, has emerged as a model organism to study genome evolution of plant pathogens. Here, we compared genomes of Z. tritici isolates and of sister species infecting wild grasses to address the evolution of genome composition and structure., Results: Using long-read technology, we sequenced and assembled genomes of Z. ardabiliae, Z. brevis, Z. pseudotritici and Z. passerinii, together with two isolates of Z. tritici. We report a high extent of genome collinearity among Zymoseptoria species and high conservation of genomic, transcriptomic and epigenomic signatures of compartmentalization. We identify high gene content variability both within and between species. In addition, such variability is mainly limited to the accessory chromosomes and accessory compartments. Despite strong host specificity and non-overlapping host-range between species, predicted effectors are mainly shared among Zymoseptoria species, yet exhibiting a high level of presence-absence polymorphism within Z. tritici. Using in planta transcriptomic data from Z. tritici, we suggest different roles for the shared orthologs and for the accessory genes during infection of their hosts., Conclusion: Despite previous reports of high genomic plasticity in Z. tritici, we describe here a high level of conservation in genomic, epigenomic and transcriptomic composition and structure across the genus Zymoseptoria. The compartmentalized genome allows the maintenance of a functional core genome co-occurring with a highly variable accessory genome.

Barley mlo mutants are well known for their profound resistance against powdery mildew disease. Recently, mlo mutant plants were generated in hexaploid bread wheat ( Triticum aestivum ) with the help of transgenic (transcription-activator-like nuclease, TALEN) and non-transgenic (targeted induced local lesions in genomes, TILLING) biotechnological approaches. While full-gene knockouts in the three wheat Mlo ( TaMlo ) homoeologs, created via TALEN, confer full resistance to the wheat powdery mildew pathogen ( Blumeria graminis f.sp. tritici ), the currently available TILLING-derived Tamlo missense mutants provide only partial protection against powdery mildew attack. Here, we studied the infection phenotypes of TALEN- and TILLING-derived Tamlo plants to the two hemibiotrophic pathogens Zymoseptoria tritici , causing Septoria leaf blotch in wheat, and Magnaporthe oryzae pv. Triticum ( MoT ), the causal agent of wheat blast disease. While Tamlo plants showed unaltered outcomes upon challenge with Z. tritici , we found evidence for allele-specific levels of enhanced susceptibility to MoT , with stronger powdery mildew resistance correlated with more invasive growth by the blast pathogen. Surprisingly, unlike barley mlo mutants, young wheat mlo mutant plants do not show undesired pleiotropic phenotypes such as spontaneous callose deposits in leaf mesophyll cells or signs of early leaf senescence. In conclusion, our study provides evidence for allele-specific levels of enhanced susceptibility of Tamlo plants to the hemibiotrophic wheat pathogen MoT .

Zymoseptoria tritici is a filamentous fungus causing Septoria tritici blotch in wheat. The pathogen has a narrow host range and infections of grasses other than susceptible wheat are blocked early after stomatal penetration. During these abortive infections, the fungus shows a markedly different gene expression pattern. However, the underlying mechanisms causing differential gene expression during host and non-host interactions are largely unknown, but likely include transcriptional regulators responsible for the onset of an infection programme in compatible hosts. MoCOD1, a member of the fungal Zn(II) 2 Cys 6 transcription factor family, has been shown to directly affect pathogenicity in the rice blast pathogen Magnaporthe oryzae. Here, we analyse the role of the putative transcription factor Zt107320, a homologue of MoCOD1, during infection of compatible and incompatible hosts by Z. tritici. We show for the first time that Zt107320 is differentially expressed in host versus non-host infections and that lower expression corresponds to an incompatible infection of non-hosts. Applying reverse genetics approaches, we further show that Zt107320 regulates the dimorphic switch as well as the growth rate of Z. tritici and affects fungal cell wall composition in vitro. Moreover, ∆Zt107320 mutants showed reduced virulence during compatible infections of wheat. We conclude that Zt107320 directly influences pathogen fitness and propose that Zt107320 is involved in the regulation of growth processes and pathogenicity during infection., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Meiosis is a key cellular process of sexual reproduction that includes pairing of homologous sequences. In many species however, meiosis can also involve the segregation of supernumerary chromosomes, which can lack a homolog. How these unpaired chromosomes undergo meiosis is largely unknown. In this study we investigated chromosome segregation during meiosis in the haploid fungus Zymoseptoria tritici that possesses a large complement of supernumerary chromosomes. We used isogenic whole chromosome deletion strains to compare meiotic transmission of chromosomes when paired and unpaired. Unpaired chromosomes inherited from the male parent as well as paired supernumerary chromosomes in general showed Mendelian inheritance. In contrast, unpaired chromosomes inherited from the female parent showed non-Mendelian inheritance but were amplified and transmitted to all meiotic products. We concluded that the supernumerary chromosomes of Z. tritici show a meiotic drive and propose an additional feedback mechanism during meiosis, which initiates amplification of unpaired female-inherited chromosomes., Competing Interests: MH, GK, EH No competing interests declared, (© 2018, Habig et al.)

The haploid genome of the pathogenic fungus Zymoseptoria tritici is contained on "core" and "accessory" chromosomes. While 13 core chromosomes are found in all strains, as many as eight accessory chromosomes show presence/absence variation and rearrangements among field isolates. The factors influencing these presence/absence polymorphisms are so far unknown. We investigated chromosome stability using experimental evolution, karyotyping, and genome sequencing. We report extremely high and variable rates of accessory chromosome loss during mitotic propagation in vitro and in planta Spontaneous chromosome loss was observed in 2 to >50% of cells during 4 weeks of incubation. Similar rates of chromosome loss in the closely related Zymoseptoria ardabiliae suggest that this extreme chromosome dynamic is a conserved phenomenon in the genus. Elevating the incubation temperature greatly increases instability of accessory and even core chromosomes, causing severe rearrangements involving telomere fusion and chromosome breakage. Chromosome losses do not affect the fitness of Zymoseptoria tritici in vitro , but some lead to increased virulence, suggesting an adaptive role of this extraordinary chromosome instability., (Copyright © 2018 by the Genetics Society of America.)

The fungal wheat pathogen Zymoseptoria tritici possesses a large complement of accessory chromosomes showing presence/absence polymorphism among isolates. These chromosomes encode hundreds of genes; however, their functional role and why the chromosomes have been maintained over long evolutionary times are so far not known. In this study, we addressed the functional relevance of eight accessory chromosomes in reference isolate IPO323. We induced chromosome losses by inhibiting the β-tubulin assembly during mitosis using carbendazim and generated several independent isogenic strains, each lacking one of the accessory chromosomes. We confirmed chromosome losses by electrophoretic karyotyping and whole-genome sequencing. To assess the importance of the individual chromosomes during host infection, we performed in planta assays comparing disease development results in wild-type and chromosome mutant strains. Loss of the accessory chromosomes 14, 16, 18, 19, and 21 resulted in increased virulence on wheat cultivar Runal but not on cultivars Obelisk, Titlis, and Riband. Moreover, some accessory chromosomes affected the switch from biotrophy to necrotrophy as strains lacking accessory chromosomes 14, 18, 19, and 21 showed a significantly earlier onset of necrosis than the wild type on the Runal cultivar. In general, we observed that the timing of the lifestyle switch affects the fitness of Z. tritici Taking the results together, this study was the first to use a forward-genetics approach to demonstrate a cultivar-dependent functional relevance of the accessory chromosomes of Z. tritici during host infection. IMPORTANCE Zymoseptoria tritici is a prominent fungal pathogen of wheat of worldwide distribution. This fungus shows a remarkable genome organization, with a large number of chromosomes that are present in only some isolates and therefore considered to be "accessory" chromosomes. To date, the function of these accessory chromosomes in Z. tritici has been unknown, although their maintenance in the species over evolutionary times suggests a functional relevance. Here we deleted whole accessory chromosomes to test the effect of these chromosomes on host specificity and virulence of the fungus. We show for the first time that some accessory chromosomes of Z. tritici affect the fitness of the fungus during host infection in a cultivar-dependent manner. These results show that the accessory chromosomes encode host-specific virulence determinants having a negative effect on fitness. Understanding the population dynamic of the accessory chromosomes and the molecular interaction of pathogen and plant traits is crucial to improve wheat-breeding strategies., (Copyright © 2017 Habig et al.)

Pathogens represent a universal threat to other living organisms. Most organisms express antimicrobial proteins and peptides, such as lysozymes, as a protection against these challenges. The nematode Caenorhabditis elegans harbours 15 phylogenetically diverse lysozyme genes, belonging to two distinct types, the protist- or Entamoeba-type (lys genes) and the invertebrate-type (ilys genes) lysozymes. In the present study we characterized the role of several protist-type lysozyme genes in defence against a nematocidal strain of the Gram-positive bacterium Bacillus thuringiensis. Based on microarray and subsequent qRT-PCR gene expression analysis, we identified protist-type lysozyme genes as one of the differentially transcribed gene classes after infection. A functional genetic analysis was performed for three of these genes, each belonging to a distinct evolutionary lineage within the protist-type lysozymes (lys-2, lys-5, and lys-7). Their knock-out led to decreased pathogen resistance in all three cases, while an increase in resistance was observed when two out of three tested genes were overexpressed in transgenic lines (lys-5, lys-7, but not lys-2). We conclude that the lysozyme genes lys-5, lys-7, and possibly lys-2 contribute to resistance against B. thuringiensis, thus highlighting the particular role of lysozymes in the nematode's defence against pathogens.

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.

Ceramide kinase (CerK) produces the bioactive lipid ceramide-1-phosphate (C1P) and appears as a key enzyme for controlling ceramide levels. In this study, we discovered and characterized adamantane-1-carboxylic acid (2-benzoylamino-benzothiazol-6-yl)amide (NVP-231), a potent, specific, and reversible CerK inhibitor that competitively inhibits binding of ceramide to CerK. NVP-231 is active in the low nanomolar range on purified as well as cellular CerK and abrogates phosphorylation of ceramide, resulting in decreased endogenous C1P levels. When combined with another ceramide metabolizing inhibitor, such as tamoxifen, NVP-231 synergistically increased ceramide levels and reduced cell growth. Therefore, NVP-231 represents a novel and promising compound for controlling ceramide metabolism that may provide insight into CerK physiological function.

The diversity of endosymbiotic bacteria that kill male host offspring during embryogenesis and their frequencies in certain groups of host taxa suggest that the evolution of male killing and the subsequent spread of male-killing symbionts are primarily determined by host life history characteristics. We studied the 10-spot ladybird beetle, Adalia decempunctata L. (Coleoptera: Coccinellidae), in which male killing has not been recorded previously, to test this hypothesis, and we also assessed the evolution of the male killer identified by DNA sequence analysis. Our results show that A. decempunctata harbors male-killing Rickettsia (alpha-proteobacteria). Male-killing bacteria belonging to the genus Rickettsia have previously been reported only for the congeneric two-spot ladybird beetle, Adalia bipunctata L. Phylogenetic analysis of Rickettsia DNA sequences isolated from different populations of the two host species revealed a single origin of male killing in the genus Rickettsia. The data also indicated possible horizontal transfer of symbionts between host species. In addition, A. bipunctata is known to bear at least four different male-killing symbionts in its geographic range two of which coexist in the two locations from which A. decempunctata specimens were obtained for the present study. Since only a single male-killing taxon was found in A. decempunctata, we assume that the two closely related ladybird beetle species must differ in the number and/or geographic distribution of male killers. We discuss the importance of these findings to our understanding of the evolution and dynamics of symbiotic associations between male-killing bacteria and their insect hosts.