Frequent horizontal chromosome transfer between asexual fungal insect pathogens.

Entire chromosomes are typically only transmitted vertically from one generation to the next. The horizontal transfer of such chromosomes has long been considered improbable, yet gained recent support in several pathogenic fungi where it may affect the fitness or host specificity. To date, it is unknown how these transfers occur, how common they are, and whether they can occur between different species. In this study, we show multiple independent instances of horizontal transfers of the same accessory chromosome between two distinct strains of the asexual entomopathogenic fungus Metarhizium robertsii during experimental co-infection of its insect host, the Argentine ant. Notably, only the one chromosome-but no other-was transferred from the donor to the recipient strain. The recipient strain, now harboring the accessory chromosome, exhibited a competitive advantage under certain host conditions. By phylogenetic analysis, we further demonstrate that the same accessory chromosome was horizontally transferred in a natural environment between M. robertsii and another congeneric insect pathogen, 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.

Combined pangenomics and transcriptomics reveals core and redundant virulence processes in a rapidly evolving fungal plant pathogen.

BACKGROUND: Studying genomic variation in rapidly evolving pathogens potentially enables identification of genes supporting their "core biology", being present, functional and expressed by all strains or "flexible biology", varying between strains. Genes supporting flexible biology may be considered to be "accessory", whilst the "core" gene set is likely to be important for common features of a pathogen species biology, including virulence on all host genotypes. The wheat-pathogenic fungus Zymoseptoria tritici represents one of the most rapidly evolving threats to global food security and was the focus of this study. RESULTS: We constructed a pangenome of 18 European field isolates, with 12 also subjected to RNAseq transcription profiling during infection. Combining this data, we predicted a "core" gene set comprising 9807 sequences which were (1) present in all isolates, (2) lacking inactivating polymorphisms and (3) expressed by all isolates. A large accessory genome, consisting of 45% of the total genes, was also defined. We classified genetic and genomic polymorphism at both chromosomal and individual gene scales. Proteins required for essential functions including virulence had lower-than average sequence variability amongst core genes. Both core and accessory genomes encoded many small, secreted candidate effector proteins that likely interact with plant immunity. Viral vector-mediated transient in planta overexpression of 88 candidates failed to identify any which induced leaf necrosis characteristic of disease. However, functional complementation of a non-pathogenic deletion mutant lacking five core genes demonstrated that full virulence was restored by re-introduction of the single gene exhibiting least sequence polymorphism and highest expression. CONCLUSIONS: These data support the combined use of pangenomics and transcriptomics for defining genes which represent core, and potentially exploitable, weaknesses in rapidly evolving pathogens.

Non-Mendelian transmission of accessory chromosomes in fungi.

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.

Apicidin biosynthesis is linked to accessory chromosomes in Fusarium poae isolates.

BACKGROUND: Fusarium head blight is a disease of global concern that reduces crop yields and renders grains unfit for consumption due to mycotoxin contamination. Fusarium poae is frequently associated with cereal crops showing symptoms of Fusarium head blight. While previous studies have shown F. poae isolates produce a range of known mycotoxins, including type A and B trichothecenes, fusarins and beauvericin, genomic analysis suggests that this species may have lineage-specific accessory chromosomes with secondary metabolite biosynthetic gene clusters awaiting description. METHODS: We examined the biosynthetic potential of 38 F. poae isolates from Eastern Canada using a combination of long-read and short-read genome sequencing and untargeted, high resolution mass spectrometry metabolome analysis of extracts from isolates cultured in multiple media conditions. RESULTS: A high-quality assembly of isolate DAOMC 252244 (Fp157) contained four core chromosomes as well as seven additional contigs with traits associated with accessory chromosomes. One of the predicted accessory contigs harbours a functional biosynthetic gene cluster containing homologs of all genes associated with the production of apicidins. Metabolomic and genomic analyses confirm apicidins are produced in 4 of the 38 isolates investigated and genomic PCR screening detected the apicidin synthetase gene APS1 in approximately 7% of Eastern Canadian isolates surveyed. CONCLUSIONS: Apicidin biosynthesis is linked to isolate-specific putative accessory chromosomes in F. poae. The data produced here are an important resource for furthering our understanding of accessory chromosome evolution and the biosynthetic potential of F. poae.

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

BACKGROUND: The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. RESULTS: Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications. CONCLUSION: The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

The differential virulence of Fusarium strains causing corneal infections and plant diseases is associated with accessory chromosome composition.

Fusarium oxysporum is a cross-kingdom pathogen. While some strains cause disseminated fusariosis and blinding corneal infections in humans, others are responsible for devastating vascular wilt diseases in plants. To better understand the distinct adaptations of F. oxysporum to animal or plant hosts, we conducted a comparative phenotypic and genetic analysis of two strains: MRL8996 (isolated from a keratitis patient) and Fol4287 (isolated from a wilted tomato [Solanum lycopersicum]). In vivo infection of mouse corneas and tomato plants revealed that, while both strains cause symptoms in both hosts, MRL8996 caused more severe corneal ulceration and perforation in mice, whereas Fol4287 induced more pronounced wilting symptoms in tomato. In vitro assays using abiotic stress treatments revealed that the human pathogen MRL8996 was better adapted to elevated temperatures, whereas the plant pathogen Fol4287 was more tolerant of osmotic and cell wall stresses. Both strains displayed broad resistance to antifungal treatment, with MRL8996 exhibiting the paradoxical effect of increased tolerance to higher concentrations of the antifungal caspofungin. We identified a set of accessory chromosomes (ACs) and protein-encoding genes with distinct transposon profiles and functions, respectively, between MRL8996 and Fol4287. Interestingly, ACs from both genomes also encode proteins with shared functions, such as chromatin remodeling and post-translational protein modifications. Our phenotypic assays and comparative genomics analyses lay the foundation for future studies correlating genotype with phenotype and for developing targeted antifungals for agricultural and clinical uses.

Accessory Chromosome-Acquired Secondary Metabolism in Plant Pathogenic Fungi: The Evolution of Biotrophs Into Host-Specific Pathogens.

Accessory chromosomes are strain- or pathotype-specific chromosomes that exist in addition to the core chromosomes of a species and are generally not considered essential to the survival of the organism. Among pathogenic fungal species, accessory chromosomes harbor pathogenicity or virulence factor genes, several of which are known to encode for secondary metabolites that are involved in plant tissue invasion. Accessory chromosomes are of particular interest due to their capacity for horizontal transfer between strains and their dynamic "crosstalk" with core chromosomes. This review focuses exclusively on secondary metabolism (including mycotoxin biosynthesis) associated with accessory chromosomes in filamentous fungi and the role accessory chromosomes play in the evolution of secondary metabolite gene clusters. Untargeted metabolomics profiling in conjunction with genome sequencing provides an effective means of linking secondary metabolite products with their respective biosynthetic gene clusters that reside on accessory chromosomes. While the majority of literature describing accessory chromosome-associated toxin biosynthesis comes from studies of Alternaria pathotypes, the recent discovery of accessory chromosome-associated biosynthetic genes in Fusarium species offer fresh insights into the evolution of biosynthetic enzymes such as non-ribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs) and regulatory mechanisms governing their expression.

Histone modifications rather than the novel regional centromeres of Zymoseptoria tritici distinguish core and accessory chromosomes.

BACKGROUND: Supernumerary chromosomes have been found in many organisms. In fungi, these "accessory" or "dispensable" chromosomes are present at different frequencies in populations and are usually characterized by higher repetitive DNA content and lower gene density when compared to the core chromosomes. In the reference strain of the wheat pathogen, Zymoseptoria tritici, eight discrete accessory chromosomes have been found. So far, no functional role has been assigned to these chromosomes; however, they have existed as separate entities in the karyotypes of Zymoseptoria species over evolutionary time. In this study, we addressed what-if anything-distinguishes the chromatin of accessory chromosomes from core chromosomes. We used chromatin immunoprecipitation combined with high-throughput sequencing ("ChIP-seq") of DNA associated with the centromere-specific histone H3, CENP-A (CenH3), to identify centromeric DNA, and ChIP-seq with antibodies against dimethylated H3K4, trimethylated H3K9 and trimethylated H3K27 to determine the relative distribution and proportion of euchromatin, obligate and facultative heterochromatin, respectively. RESULTS: Centromeres of the eight accessory chromosomes have the same sequence composition and structure as centromeres of the 13 core chromosomes and they are of similar length. Unlike those of most other fungi, Z. tritici centromeres are not composed entirely of repetitive DNA; some centromeres contain only unique DNA sequences, and bona fide expressed genes are located in regions enriched with CenH3. By fluorescence microscopy, we showed that centromeres of Z. tritici do not cluster into a single chromocenter during interphase. We found dramatically higher enrichment of H3K9me3 and H3K27me3 on the accessory chromosomes, consistent with the twofold higher proportion of repetitive DNA and poorly transcribed genes. In contrast, no single histone modification tested here correlated with the distribution of centromeric nucleosomes. CONCLUSIONS: All centromeres are similar in length and composed of a mixture of unique and repeat DNA, and most contain actively transcribed genes. Centromeres, subtelomeric regions or telomere repeat length cannot account for the differences in transfer fidelity between core and accessory chromosomes, but accessory chromosomes are greatly enriched in nucleosomes with H3K27 trimethylation. Genes on accessory chromosomes appear to be silenced by trimethylation of H3K9 and H3K27.