27Chromosome and genome stability are important for normal cell function as instability often 28 correlates with disease and dysfunction of DNA repair mechanisms. Many organisms maintain 29 supernumerary or accessory chromosomes that deviate from standard chromosomes. The 30 pathogenic fungus Zymoseptoria tritici has as many as eight accessory chromosomes, which are 31 highly unstable during meiosis and mitosis, transcriptionally repressed, show enrichment of 32 repetitive elements, and enrichment with heterochromatic histone methylation marks, e.g., 33trimethylation of H3 lysine 9 or lysine 27 (H3K9me3, H3K27me3). To elucidate the role of 34 heterochromatin on genome stability in Z. tritici, we deleted the genes encoding the 35 methyltransferases responsible for H3K9me3 and H3K27me3, kmt1 and kmt6, respectively, and 36 generated a double mutant. We combined experimental evolution and genomic analyses to 37 determine the impact of these deletions on chromosome and genome stability, both in vitro and 38 in planta. We used whole genome sequencing, ChIP-seq, and RNA-seq to compare changes in 39 genome and chromatin structure, and differences in gene expression between mutant and 40 wildtype strains. Analyses of genome and ChIP-seq data in H3K9me3-deficient strains revealed 41 dramatic chromatin reorganization, where H3K27me3 is mostly relocalized into regions that are 42 enriched with H3K9me3 in wild type. Many genome rearrangements and formation of new 43 chromosomes were found in the absence of H3K9me3, accompanied by activation of transposable 44 elements. In stark contrast, loss of H3K27me3 actually increased the stability of accessory 45 chromosomes under normal growth conditions in vitro, even without large scale changes in gene 46 activity. We conclude that H3K9me3 is important for the maintenance of genome stability 47 because it disallows H3K27me3 in these regions. In this system, H3K27me3 reduces the overall 48 stability of accessory chromosomes, generating a "metastable" state for these quasi-essential 49 regions of the genome. 50 3 Author Summary 51 Genome and chromosome stability are essential to maintain normal cell function and viability. 52However, differences in genome and chromosome structure are frequently found in organisms 53 that undergo rapid adaptation to changing environmental conditions, and in humans are often 54 found in cancer cells. We study genome instability in a fungal pathogen that exhibits a high degree 55 of genetic diversity. Regions that show extraordinary diversity in this pathogen are the 56 transposon-rich accessory chromosomes, which contain few genes that are of unknown benefit 57 to the organism but maintained in the population and thus considered "quasi essential". 58Accessory chromosomes in all fungi studied so far are enriched with markers for 59 heterochromatin, namely trimethylation of H3 lysine 9 and 27 (H3K9me3, H3K27me3). We show 60 that loss of these heterochromatin marks has strong but opposing effects on genome stability. 61While loss of the transposon-associate...