The dim2 DNA methyltransferase gene is functional in several Z. tritici isolates

Based on previous findings reporting a recent inactivation of dim2 in the Z. tritici reference isolate IPO323 [25] we carefully inspected the genomic locus in different isolates to characterize the extent of mutational events in different genomes. We identified a non-truncated dim2 gene in an Iranian Z. tritici isolate, Zt10, and set out to investigate its recent evolution in the Zymoseptoria species complex in a much larger collection of genome sequences than previously [25] available. We used BLAST to search for the sequence of dim2 in 22 high-quality assemblies obtained by SMRT sequencing of Z. tritici (Fig 1) [32–35], as well as 17 genomes of Z. ardabiliae, and nine genomes of Z. brevis (S1 Table), both closely related sister species of Z. tritici and considered to be endemic to Iran. We detected a single dim2 homolog in each of the Z. ardabiliae and Z. brevis genomes but found multiple mutated and presumably non-functional copies of dim2 in 17 out of 22 Z. tritici genomes (Fig 1). Five Z. tritici isolates contained one presumably functional, non-mutated copy of dim2, often in addition to multiple mutated, non-functional copies, except for isolate Zt469, which contained one functional dim2 gene and no additional copies. For the purpose of this study, we consider dim2 alleles intact and ‘functional’ when they do not contain pre-mature stop codons or frameshift mutations, even though some SNVs may generate missense mutations. In contrast, ‘non-functional’ alleles contain numerous mutations including pre-mature stop codons that are predicted to abrogate protein function. Actual methylation activity was tested in selected strains by assaying cytosine methylation in vivo.

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Fig 1. Overview of sampling location, year, host plant, and presence of a functional dim2 DNA methyltransferase gene of Z. tritici isolates analyzed in this study.

Aegilops sp., wild grass in Triticeae; Triticum aestivum, bread wheat; T. turgidum durum, Durum wheat. *non-functional but only recently mutated native dim2 gene.

https://doi.org/10.1371/journal.pgen.1009448.g001

Zt469 was isolated from Aegilops sp., all other isolates were collected from wheat. Four of five isolates with functional dim2 are from Iran (Zt10, Zt11, Zt289, Zt469) [32,36], and one is from Tunisia (TN09, collected from durum wheat) [32]. We further tested whether presence of a non-mutated dim2 allele is commonly found among Iranian isolates. Twelve additional isolates from Iran (S1 Table) were assayed by PCR and Sanger sequencing and in all cases, we confirmed presence of an intact dim2 allele. A region of chromosome 6 of IPO323 had been previously identified as the native dim2 locus; all additional, non-functional copies are located in subtelomeric regions embedded in repetitive DNA [25]. Consistent with this idea, we found that all non-mutated dim2 genes were located at the predicted native locus on chromosome 6 (Fig 2A and S2 Table). Throughout this study, we refer to dim2 in the original locus on chromosome 6, functional or non-functional, as the ‘native’ gene or allele; dim2 copies outside the native locus are referred to as ‘non-native’ or ‘additional’ alleles.

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Fig 2. Sequence comparisons of dim2 alleles in Z.

tritici isolates. (A) Mutations of dim2 in original and ‘deRIPed’ alleles identified by alignment to the functional allele of Zt469. Shown are all native alleles (located on chromosome 6) and one representative non-native, additional copy per isolate. Native alleles (functional and non-functional) lack mutations in the DNA methyltransferase domain that are present in all non-native alleles, except for isolates OregS90 and CRI10. (B) Pairwise sequence comparison of the native, intact dim2 allele from Zt469 with all full-length native and additional dim2 sequences (see S2 Table). No additional dim2 copies were detected in the genome of Zt469 (from Aegilops sp.) whereas all other isolates (from Triticum spp.) have additional copies of dim2. Non-native, additional copies (black dots) in the isolates with functional or recently mutated dim2 (except for UR95) showed higher sequence diversity compared to those with inactivated dim2, where alleles at the native locus (yellow triangle) are amongst the more diverged copies. This supports the idea that this is the ancestral, now heavily mutated allele of dim2. Alleles or isolates are arranged in the same order in panels A (top to bottom) and B (left to right).

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Analysis of Z. tritici dim2 alleles reveals multiple cycles of recombination and inactivation

Previously, gene amplification of the native dim2 allele followed by RIP had been suggested as the most likely cause for the apparent lack of 5mC in the Z. tritici IPO323 genome [25,37]. We here conducted a detailed analysis of mutations within native and non-native dim2 genes in the Z. tritici genomes and found some unexpected patterns.

As previously reported, non-functional copies of dim2 contain numerous transition mutations as a consequence of RIP, thus to compare sequences of native and additional (non-native) copies we “deRIPed” mutant copies by reverting C→T and G→A transitions using the functional dim2 from Zt469 as reference, because it likely represents one ancestral allele. We found that all non-native alleles share a specific pattern of transition and, surprisingly, transversion mutations in the DNA methyltransferase domain that are not found in the vast majority of native dim2 genes (Figs 2A and S1A and S2). Transversions are not a consequence of RIP [38,39], thus a mixture of transitions, transversions, and deletions suggests that dim2 was mutated not simply as a result of amplification of the native gene followed by widespread RIP, as originally proposed [25]. Rather, integration of a different dim2 allele (hereafter called ‘transversion allele’) of unknown origin into subtelomeric regions is likely to have resulted in subsequent gene amplification, RIP and presence of multiple non-functional, non-native dim2 copies. The presence of the same transversion mutations in all additional copies indicates that this allele may have resulted from a single integration event in an ancestral Z. tritici isolate. In support of this hypothesis, we found that the flanking regions of the additional, but not the native, copies share homology and are annotated as retrotransposons, suggesting that integration and amplification of non-native copies may have been TE-mediated. The TEs that are found in the flanking regions of most of the additional dim2 copies belong to the LINE family.

While this idea explains events at subtelomeric integrations sites, we also identified two isolates in which the native dim2 gene on chromosome 6 shares the specific transversion mutations in the DNA methyltransferase domain, OregS90 and CRI10 (from the USA and Czech Republic, respectively) (Fig 2A). The native OregS90 dim2 is truncated and surrounded by TEs and the native dim2 of CRI10 lacks mutations in the 3’ region that are present in all additional, but not native, copies.

Five native dim2 genes have a shared deletion in the 5’ region that is not found in any of the additional copies, and a 27-bp insertion at the end of the reading frame. The 5’ deletion results in a 99–109 amino acid in-frame deletion and not in any known functionally important domains, while the ill-conserved C-terminus is extended by nine amino acids (S3 Fig). These results suggest that distinct functional dim2 alleles are present, and given the previously identified signatures of introgression in Z. tritici genomes [31] we consider that these different dim2 alleles at the native locus may reflect separate integration events that have been directed by sequence identity. In summary, our analyses revealed that gene amplification followed by RIP alone is insufficient to explain the distribution of variant dim2 alleles in Z. tritici isolates.

To further investigate the evolutionary history of dim2 in Z. tritici isolates, we compared the sequence identity of the non-mutated dim2 gene from Zt469 to all other, native and additional, copies of dim2 in the 22 Z. tritici genomes (Fig 2B). Gene amplification followed by RIP predicts that by accumulating transition mutations all copies should become quite similar to each other; RIP becomes less efficient or stops when sequence identity between alleles drops to ~80% [38]. Instead, our analyses revealed that 18/21 isolates showed two distinct patterns, the first matching the prediction because isolates lacking functional dim2 showed higher levels of DNA sequence identities (85–95%) between native and additional alleles (Fig 2B and S2 Table; red, “non-functional”, pattern 1). Here, all copies have suffered repeated RIP cycles, and all copies are non-functional. When, however, a functional, native dim2 allele is present, the additional copies show lower DNA sequence identities (84–88%), likely reducing RIP efficiency and preventing the functional allele from being inactivated. The functional alleles are almost identical (>96%) to the Zt469 allele (Fig 2B and S2 Table; dark blue, “intact”, pattern 2; Zt10, Zt289, Zt11, TN09). This second pattern was unexpected; the most parsimonious explanation is replacement of non-functional alleles at the native locus with novel dim2 alleles, perhaps by recombination with wild grass infecting isolates, such as Zt469. Introgression events between Zymoseptoria species have recently been described [31] and wild grass infecting Z. tritici isolates, sister species and wheat infecting Z. tritici populations co-exist in the same geographical regions in the Middle East [40].

We discerned a third group of three isolates, which suggests that repeated cycles of transposon- or recombination-mediated amplification of dim2 alleles are still ongoing (Fig 2B and S2 Table; light blue, “recently inactivated”, pattern 3; YEQ92, ISY92, UR95). YEQ92 and ISY92 contain non-functional dim2 alleles at the native locus that are still similar (96 and 92%, respectively) to the functional dim2, suggesting a more recent inactivation of dim2 in these isolates. All additional, subtelomeric copies have low sequence identities as in the second pattern described above. UR95 has two recently mutated dim2 alleles, one at the native locus (Zt469 allele) and one in the subtelomeric region on chromosome 8 (transversion allele); both are non-functional alleles but contain relatively few mutations and are still similar to the functional dim2 allele (>90%) suggesting recent integration and inactivation. In summary, our findings suggest that there are multiple functional alleles of dim2 at the native locus present in the Z. tritici population. One group of alleles is derived from the Zt469 allele, a second group contains the “fingerprint” of the transversion allele when compared to the Zt469 allele and a third group contains the 5’ deletion. All alleles exhibit some degree of variation in terms of synonymous, non-synonymous mutations, and INDELs but none of these mutations affect sites that are known to be essential for protein function indicating that all of these alleles may likely encode functional proteins (S3 Fig).

Integration of a functional dim2 allele at the native locus restores 5mC in Zt09

To assess the role of dim2 in DNA methylation we generated mutants in which we either restored or deleted dim2. We integrated the functional dim2 gene of the Iranian Zt10 isolate into the native dim2 locus of Zt09 (derived from reference isolate IPO323) by selecting for hygromycin resistance, conferred by the hph gene, generating the strain Zt09::dim2. In Zt10, we replaced the functional dim2 gene with hph, generating the strain Zt10Δdim2. Integration or deletion of the dim2 alleles were verified by Southern analyses (S4 Fig). To test whether dim2 affects growth or infection, we compared the mutant and respective wild-type strains under different in vitro conditions as well as during host infection. We did not detect any differences between wild-type and mutant strains in vitro (S5 Fig) but integration of the functional dim2 in Zt09 resulted in a small but significant reduction in necrosis and asexual fruiting body formation (measured as number of asexual fruiting bodies, pycnidia, per leaf area) in planta (S6 Fig). We therefore conclude that presence of dim2 is dispensable under the tested in vitro conditions and that re-integration of dim2 into the Zt09 background decreases virulence in planta. We note that restoring DNA methylation in Zt09 after absence of dim2 for likely thousands of generations does not necessarily reflect the influence that DNA methylation may have in Z. tritici under normal conditions. Deletion of dim2 in Zt10 did not have significant immediate impact on virulence; this needs to be further addressed in long term studies.

To validate the presence of 5mC and to compare patterns between isolates and mutants, we performed whole-genome bisulfite sequencing (WGBS). We sequenced three replicates of each wild-type isolate (Zt09 and Zt10) and mutant strain (Zt09::dim2 and Zt10Δdim2). As a control for the bisulfite conversion rate, we added Lambda DNA to each sample; based on this, the bisulfite conversion rate was >99.5%. To confirm the WGBS sequencing results, we performed an additional independent bisulfite conversion, amplified target regions by PCR, cloned and sequenced the fragments, and we performed Southern blot analyses using 5mC-sensitive and -insensitive restriction enzymes (S7 Fig).

We detected high numbers of methylated cytosines in isolates with functional dim2 alleles, i.e., the wild-type Iranian isolate Zt10 and Zt09::dim2. Surprisingly, we also detected low levels of 5mC in the absence of dim2, i.e., in Zt10Δdim2 and Zt09, which had been missed by mass spectrometry in earlier studies [25]. While on average ~300,000 sites in Zt10 and ~400,000 sites in Zt09::dim2 showed 5mC in the presence of dim2, only ~12,000 (Zt09) and ~40,000 (Zt10Δdim2) 5mC sites were found when dim2 was absent (Fig 3A). In all strains, 5mC was restricted to previously annotated TEs and adjacent non-coding regions (Fig 3A). We did not find evidence for gene body or promoter methylation, except for few TE-derived genes. By correlating the 5mC data with previously published histone methylation maps [41] we found that 5mC co-localizes with H3K9me3 marks (Fig 3B). This finding is consistent with observations in N. crassa, C. neoformans, and Arabidopsis thaliana [21,42–45].

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Fig 3. Localization and site preferences of cytosine methylation in Z.

tritici genomes in presence and absence of dim2. (A) Number of 5mC methylated sites detected by whole genome bisulfite sequencing (WGBS) in TEs, intergenic regions, or genes. The vast majority of methylated sites are localized in TEs followed by intergenic regions and TE-derived genes. (B) 5mC co-localizes with H3K9me3 and TEs in Zt09. Shown are ChIP-seq tracks for H3K4me2, H3K27me3 and H3K9me3 [41], and 5mC sites detected in a representative region on chromosome 9 in the genomes of Zt09 and Zt09::dim2 (three replicates). (C) Genome-wide 5mC levels in the different isolates and respective mutants. 5mC levels are higher in the presence of a functional dim2, and dim2 is required for all non-CG methylation. (D) 5mC levels and site preferences in TEs. CG sites are preferred, followed by CA, and CC. 5mC is almost completely absent from CTs.

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We next analyzed the specific context of DNA methylation. In strains without functional dim2, we found 5mC exclusively in CG contexts (genome wide ~0.2% in Zt09 and ~1% in Zt10Δdim2; percentage based on all Cs in the genome). In the presence of dim2, cytosines in CG as well as CHG and CHH contexts were methylated. Genome wide, ~5 or 6% of CGs and <1% of CHGs or CHHs were methylated in Zt10 or Zt09::dim2, respectively (Fig 3C). In sequences annotated as TEs, however, up to 60% of all CGs were methylated (Fig 3D). Methylation of CGs occurred most frequently (~43%, ~61%), but we also detected methylation at CAs (~10%, ~22%), and to lesser extent at CCs (~3%, ~8%), in Zt10 or Zt09::dim2, respectively. At CTs, there was hardly any detectable methylation (<0.01%; Figs 3D and S8). This was surprising, as CTs are the most abundant CH dinucleotides in TEs while CAs are the least abundant sites in TEs of both Zt09 and Zt10 (S3 Table). This suggests a strong site-specific preference of Dim2 for CAs but not CTs in Z. tritici. We furthermore calculated methylation levels (number of 5mC reads/position) and found that 5mC levels and overlap of 5mC sites between replicates are highest in Zt09, where only very few CpG sites are maintained (S9 Fig). Zt10Δdim2 shows high variability of 5mC site positions and methylation levels between the three replicates (independent transformants) indicating that 5mC sites are nearly randomly and progressively lost after dim2 deletion. Zt10 and Zt09::dim2 both have comparable methylation levels that are consistent between replicates (S9 Fig).

Upon integration of a functional dim2 allele in Zt09 we found that overall 5mC levels were higher when compared to Zt10 (compare Fig 3C, left- and right-most panels) and CG methylation levels in Zt10Δdim2 were higher compared to Zt09 (compare Fig 3C, two middle panels). One possible explanation for higher methylation levels in the Zt09::dim2 strain is that upon integration of dim2, a high number of sites are methylated but then reduced by selective conditions. This may occur after several rounds of infection and prolonged growth until a balanced level of 5mC sites is achieved, as found in Zt10. It is also possible that methylation levels differ between isolates and may depend on the abundance of different TE families that vary in terms of methylation density. Our findings suggest that in the absence of the de novo DNA methyltransferase Dim2, 5mC levels decrease significantly over relatively short evolutionary time spans (< 12,000 years, after speciation of Z. tritici [36]).

DNA methylation can be maintained in absence of dim2

We next asked how 5mC can persist in the genome of Z. tritici in the absence of a functional dim2 gene. The presence of CG methylation in isolates without dim2 suggested the presence of an additional DNMT. Thus, we searched for putative DNMT coding regions in the genomes of Z. tritici, Z. ardabiliae, and Z. brevis with the conserved DNMT domain of Dim2 as query in a BLASTp search. In all genomes, we found two additional predicted DNMTs (S11 Fig). One is similar to Dnmt5 of Cryptococcus neoformans [20] (dnmt5, Zt_chr6_00685), the other is a homolog of N. crassa RID [17] (rid, Zt_chr5_00047; manually corrected gene annotation) [46]. Genes for Dnmt5 and Rid are present in all Zymoseptoria spp. genomes we analyzed. The dnmt5 alleles are highly conserved and show very little inter- or intraspecies diversity (S1B Fig). In contrast, rid shows an exceptionally high inter- and intraspecies diversity with three highly distinct alleles present in Zymoseptoria spp. (S1C Fig). This diversity may stem from introgression events between Zymoseptoria species as recently described for highly variable regions in the genome of Z. tritici [31]. However, presence of the different rid alleles does not correlate with presence of a functional dim2 gene in Z. tritici.

RID and its homolog Masc1 in Ascobolus immersus are involved in genome defense during pre-meiosis [17,18]. Although Masc1 is presumed to be a de novo DNMT acting during the sexual cycle of A. immersus [18], there are still no enzyme activity data available for either Masc1 or RID. In contrast, Dnmt5 has been characterized as a 5mCG maintenance DNMT in C. neoformans [20,21] and is thus the most likely candidate for maintenance of 5mC at CG sites that we observed here. Dnmt5 is moderately expressed during infection of wheat in both, Zt09 and Zt10, while the expression levels of rid are very low consistent with its known role in pre-meiotic, and not mitotic, stages (S10 Fig) [17]. Further studies will address the role of Dnmt5 in maintenance and impact on 5mC in Z. tritici.

Presence of dim2 impacts nucleotide composition and activity of TEs

DNA cytosine methylation is correlated with increased C→T mutation rates [47,48]. In Z. tritici and most other fungi [22], 5mC is enriched in TE sequences. To evaluate whether the presence of dim2 impacts sequence composition of TEs, we analyzed TE sequences in isolates with and without a functional dim2 gene. Therefore, we extracted TE sequences, calculated dinucleotide frequencies relative to the TE content for each genome (S3 Table), and visualized the difference in relative frequencies of specific dinucleotides in TEs in the different isolates by correspondence analysis. We found that isolates lacking functional dim2 have more CA/TG sites within TEs compared to isolates with functional dim2 (Fig 4A and 4B). Conversely, isolates with functional dim2 contain more TA sites in TEs (Fig 4A and 4B). Two isolates with recently inactivated dim2, ISY92 and YEQ92, show the same pattern as isolates with functional dim2 but UR95, with two recently inactivated copies, groups with isolates lacking functional dim2. Based on the presence of additional dim2 copies with relatively high sequence identities in UR95 (Fig 2B), we propose that integration of a functional dim2 may lead to rapid inactivation by RIP. Thus, grouping of UR95 with the ‘non-functional’ isolates can be explained by limited Dim2 activity before inactivation of the gene by RIP, a prediction that will be tested in future studies. CA/TG sites are the least frequent sites in TEs, while TA sites are the most abundant. We observed the same pattern (i.e., increased number of TA sites) when we included the sister species Z. ardabiliae (Za17) and Z. brevis (Zb87) and the more distantly related barley pathogen, Z. passerinii (Zpa63), all of which contain a single, functional dim2 (Fig 4B and S3 Table). This pattern is only detectable in TEs, not in the rest of the genome (S3 Table).

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Fig 4. Impact of Dim2 on nucleotide composition and TE activity.

(A) and (B) Correspondence analyses showing differences in dinucleotide (2mer) frequencies in TEs of different Z. tritici isolates (A) and other members of the Zymoseptoria species complex (B). TA and CA/TG dinucleotide frequencies have the highest impact on the observed differences between isolates and species, indicated by their relative positions on the dimension 1 versus dimension 2 plot. Isolates (even from different species) containing a functional dim2 (Zt10, Zt289, Zt11, Zt469, TN09, Zb87, Za17, Zpa63) and recently mutated dim2 (ISY92 and YEQ92) have higher TA content and cluster together, while isolates without functional dim2 have higher CA/TG frequencies in TE sequences. (C) Comparison of TE and GC content between different Z. tritici isolates and Zymoseptoria species with and without functional dim2. D) Number of expressed TEs during the time course of wheat infection is significantly higher in Z. tritici isolate Zt09 (non-functional dim2) compared to Zt10 (functional dim2) (Fisher’s Exact Test for Count Data, p-value < 2.2 x 10⁻¹⁶).

https://doi.org/10.1371/journal.pgen.1009448.g004

Accelerated mutation rates in TEs may impact TE activity, so we next determined the TE content of the various Z. tritici isolates and compared it to that observed in other Zymoseptoria species (Fig 4C). Isolates with functional dim2 had slightly lower TE content than those lacking dim2 (mean of ~16.2% vs. ~18.7%, respectively; Fig 4C) but TE content in other Zymoseptoria species, all with functional dim2, was higher than in many Z. tritici strains. Thus, there is no simple or clear correlation between TE content and de novo 5mC activity. GC content of TEs, however, is considerably lower in isolates with functional dim2 and two isolates with recently mutated dim2, ISY92 and YEQ92, further indicating that Dim2 impacts nucleotide composition (Fig 4C).

The TE content of a genome does not necessarily reflect the activity of TEs, as these sequences may be relics, i.e., mutated and thus inactivated TEs. We therefore assessed whether TEs in Zt09 and Zt10 contained annotated, transposon-related genes as a putative indicator for the presence of active TEs. In Zt09, 56 genes completely overlap (>90% of the sequence) with annotated TEs. Among those, we identified 30 transposon- or virus-related genes encoding for endonucleases, transposases, ribonucleases, and reverse transcriptases, or genes encoding virus-related domains (S4 Table). In contrast, we did not detect any fungal genes overlapping TEs in Zt10. We did find homologs of some of the transposon-associated genes of Zt09 in the genome of Zt10, but they all contained numerous transitions resulting in mis- and nonsense mutations. This indicates that Zt09 contains active TEs whereas homologous elements are inactivated in Zt10.

Lastly, we compared the expression of TEs in Zt09 and Zt10 during the time course of infection by analyzing previously published RNA-seq data [35]. We computed transcripts per million (TPM) originating from TEs in both the biotrophic and necrotrophic phases of infection and considered a TE expressed when TPM was > 0. While ~21% of TEs produced RNA in Zt10, ~35% of TEs were transcribed in Zt09 (Fig 4D) indicating that silencing of TE loci is less pronounced in Zt09 where dim2 is non-functional.

Dim2 promotes C→T transitions at CAs during mitosis

The difference between CA/TG and TA site abundance in genomes with and without functional dim2 suggests that the mutation rate, specifically for C→T transitions, is affected by the presence of 5mC. To address whether Dim2 plays a role in promoting these mutations, we conducted a one-year evolution experiment that included the Iranian isolate Zt10 (functional dim2) and the reference isolate IPO323 (non-functional dim2, isolate Zt09 is a derivate of IPO323). This was a mutation accumulation experiment on cells that were dividing by mitosis without competition or selection. Each ancestral isolate was propagated as 40 independent replicates. After 52 weeks (corresponding to ~1,000 mitotic cell divisions) the genomes of 40 evolved replicate strains per isolate and the progenitor isolates were sequenced to map SNPs. We found that the mutation rate in Zt10 was more than tenfold higher than in IPO323, on average ~170 vs. ~13 mutations per replicate (Fig 5A and S5 Table). The vast majority of mutations in Zt10 (> 95%) occurred in 5mC regions (S6 Table) and > 95% of all mutations were C→T transitions, while only ~32% of all mutations in IPO323 were C→T transitions. In Zt10, > 98% of the C→T transitions occurred at CAs (vs. ~21% in IPO323) (Fig 5B). This was a surprise, because the majority of methylated sites are at CGs. Our findings suggest the presence of a dim2-dependent mechanism that specifically targets and mutates CA/TG but not CGs in sequence repeats during mitosis. Such a mutator resembles the hitherto undescribed but much looked-for mitotic version of RIP.

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Fig 5. Number and site preferences of SNPs in isolates with functional (Zt10) and non-functional (IPO323*) dim2 after one year of mitotic growth.

(A) Detected number of SNPs in the 40 replicates of each isolate. The number of SNPs in Zt10 replicates is significantly higher compared to IPO323 (Wilcoxon rank sum test, ***p-value < 0.001). The vast majority of mutations are C to T transitions. (B) Sequence context of the mutations in the two tested isolates. The SNPs of all replicates were pooled for this analysis. C→T transitions are predominantly found in a CA context resulting in CA to TA (or GT to TA) mutations. CG sites, although preferentially methylated, are not mutated at high rates. *Isolate Zt09 is a derivate of IPO323.

https://doi.org/10.1371/journal.pgen.1009448.g005

Fungal isolates and growth conditions

All Zymoseptoria spp. isolates used in this study (S1 Table) were cultivated at 18°C in YMS (4 g yeast extract, 4 g malt, 4 g sucrose per 1 L, 20 g agar per L for plates) medium. Cultures for DNA extraction and plant infection experiments were inoculated directly from the -80°C glycerol stocks and grown in liquid YMS medium at 200 rpm for 5 days (DNA extractions) and in pre-cultures (3 days) and main cultures (2 days) for plant infections.

SMRT sequencing and assembly of Iranian isolates Zt289 and Zt469

High molecular weight DNA was extracted as previously described [70]. Library preparation and PacBio sequencing was performed at the Max Planck Genome Center in Cologne, Germany (https://mpgc.mpipz.mpg.de/home/) on a Pacific Biosciences Sequel II. One SMRT cell was sequenced per genome. Genome assemblies were performed as described [71]. In brief, genomes were assembled de novo using the SMRT Analysis software v.5 (Pacific Bioscience) using default and “fungal” parameters. The assembly quality was determined using Quast [72] and the assembly with the best quality used for further analyses. Genome assembly statistics are summarized in S7 Table.

Identification of TEs and analysis of TE expression

We annotated transposable elements with the REPET pipeline (https://urgi.versailles.inra.fr/Tools/REPET) [73] as described in [71]. Briefly, we identified repetitive element consensus sequences in each genome using TEdenovo following the developer’s recommendations [73]. We used each library of consensus sequences to annotate genomes using TEannot with default parameters. To evaluate TE activity we analyzed expression in planta using RNA-seq data of Z. tritici isolates Zt10 and Zt09 [35]. First, raw sequencing reads were quality filtered and trimmed using Trimmomatic (v0.36) [74] and mapped to the respective genome using hisat2 (v2.2.0) [75]. Reads were trimmed using the following parameters: LEADING:30 SLIDINGWINDOW:4:30 AVGQUAL:30 MINLEN:50. After mapping, the resulting aligned read files were used to assess read counts for each stage and replicate (with a total of two replicates per stage). The read count table was generated using TEcount function of the TEtranscript pipeline with the following parameter: -mode multi [76]. Briefly, TEtranscript pipeline counts both uniquely and multi-mapped reads mapped to annotated transposons and genes to attribute transcript abundance. A given TE was considered transcribed only if reads mapped all along the element length, in order to avoid redundancy [76]. Levels of expression were calculated using Transcript per Million (TPM) normalization which corresponds to the normalization of read counts with transposon (or gene) length per million. We considered a TE as ‘expressed’ if TPM > 0.

Sequence identification and comparison of DNA methyltransferases

All analyzed genomes and isolates used in this study are listed in S1 Table and originate from the following studies [31–36,40,49,71,77–82]. We identified homologs of dim2 using the predicted ‘deRIPed’ protein sequence of Z. tritici IPO323 [25] as a template. To compare the sequence identity between active and inactive dim2 copies in the genome, we used dim2 of isolate Zt469 as a reference and performed pairwise comparisons. To identify additional putative DNA methyltransferases, we used the DNA methyltransferase domain of the Dim2 protein as query for a BLAST search against the Z. tritici genomes. BLAST searches, phylogenetic trees and alignments were performed using Geneious ‘Blast’, Geneious ‘Alignment’ and Geneious ‘Map to reference’ (Geneious version 10.2.4 (http://www.geneious.com, [83]). A distance tree based on the nucleotide sequence of dim2, rid and dnmt5 was generated with the following settings: alignment type: global alignment with free end gaps, cost matrix: 65% similarity, genetic distance: Jukes-Cantor, tree build method: neighbor joining, outgroup: Zymoseptoria passerinii (Zpa63).

Generation of dim2 deletion and integration strains

We transformed the Z. tritici isolates Zt09 and Zt10 using an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol as previously described [84]. Briefly, we created plasmids containing the integration (pES189) or deletion (pES188) constructs using Gibson assembly [85] (S8 Table). Both constructs contained the hygromycin resistance cassette as selection marker. Plasmids were amplified in E. coli TOP10 cells and sequenced to confirm correct assembly of the constructs followed by electroporation of A. tumefaciens strain AGL1. Zymoseptoria tritici strains were transformed with the respective A. tumefaciens strains by co-incubation for 3–4 days at 18°C on induction medium. Following co-incubation, the strains were grown on selection medium containing hygromycin to select for integration of the constructs and cefotaxime to eliminate A. tumefaciens. Single Z. tritici colonies were selected, streaked out twice and the correct integration of the construct was verified by PCR and by Southern blot analyses (S4 Fig).

Bisulfite treatment and sequencing

We extracted high molecular weight DNA of three biological replicates of Zt10 and Zt09 and three independent transformants of Zt10Δdim2 and Zt09::dim2 as described previously [70]. Genomic DNA was sent to the Max Planck Genome Centre in Cologne, Germany (https://mpgc.mpipz.mpg.de/home/) for bisulfite treatment and sequencing. Lambda DNA was used as spike-in (~1%) to determine conversion efficiency. Genomic DNA was fragmented with a COVARIS S2 and an Illumina-compatible library was prepared with the NEXTflex Bisulfite Library Prep Kit for Illumina Sequencing (Bioo Scientific/PerkinElmer, Austin, TX). Bisulfite conversion was performed using the EZ DNA Methylation Gold Kit (Zymo Research, Irvine, CA). Illumina sequencing was performed on a HiSeq3000 machine with paired-end 150-nt read mode (S9 Table).

To confirm bisulfite sequencing results, we treated genomic DNA of the fungal strains and, as a control for conversion efficiency, the Universal Methylated DNA Standard (Zymo Research) (~100 ng) with bisulfite using the EZ DNA Methylation-Lightning Kit (Zymo Research) according to manufacturer’s instructions. Two representative loci per fungal strain and the human MLH1 for the control were amplified by PCR (ZymoTaq PreMix, Zymo Research) using specifically designed primers for amplification of the bisulfite-treated DNA (designed using the ‘Bisulfite Primer Seeker’ (Zymo Research), S8 Table). PCR products were cloned using the TOPO TA kit (Thermo Fisher Scientific) and Sanger sequenced (Eurofins Genomics, Ebersberg, Germany). Analysis of sequenced bisulfite-converted DNA sequences was performed using Geneious software version 10.2.4 [83].

Data analysis of whole genome bisulfite sequencing data

A list of software and input commands used in our analyses is provided in the S1 Text.

Reads were quality filtered using Trimmomatic (v0.36) [74] and subsequently mapped using Bismark (v0.20.0) [86]. Duplicate reads were removed. Methylated sites were extracted using the bismark_methylation_extractor applying the–no_overlap and CX options. We considered sites methylated, if at least 4 reads and ≥ 50% of reads supported methylation. We then extracted these sites (see S1 Text) in CG, CHG or CHH contexts for further analysis. Bedtools (v2.25.0) [87] was used to correlate methylation and genomics features.

Southern blots to detect DNA methylation and confirm dim2 mutant strains

To detect presence or absence of 5mC, we performed Southern blots according to standard protocols [88]. Genomic DNA was extracted using a standard phenol-chloroform extraction method [89]. The same amount of DNA and enzymes (BfuCI, DpnI, DpnII; 25 units, New England Biolabs, Frankfurt, Germany) was used as input for the different restriction digests to make restriction patterns comparable between enzymes and Z. tritici isolates for the detection of DNA methylation. Probes were generated with the PCR DIG labeling Mix (Roche, Mannheim, Germany) following the manufacturer’s instructions and chemiluminescent signals were detected using the GelDoc XR+ system (Bio-Rad, Munich, Germany).

Phenotypic assay in vitro

Spores were diluted in water (10⁷ cells/mL and tenfold dilution series to 1,000 cells/mL) and three μL of the spore suspension dilutions were pipetted on plates and incubated for ten days. To test for responses to different stress conditions in vitro, YMS plates containing NaCl (0.5 M and 1 M), sorbitol (1 M and 1.5 M), Congo Red (300 μg/mL and 500 μg/mL), H₂O₂ (1.5 mM and 2 mM), methyl methanesulfonate (0.01% and 0.005%) and two plates containing only YMS were prepared. All plates were incubated at 18°C, except for one of the YMS plates that was incubated at 28°C.

Phenotypic assay on wheat

Seedlings of the wheat cultivar Obelisk (Wiersum Plantbreeding BV, Winschoten, The Netherlands) were pre-germinated on wet sterile Whatman paper for four days under normal growth conditions (16 h at light intensity of ~200 μmol/m^-2s^-1 and 8 h darkness in growth chambers at 20°C with 90% humidity) followed by potting and growth for an additional seven days. Marked areas on the second leaves (30 leaves per strain) were inoculated with a spore suspension of 10⁷ cells/mL in H₂O and 0.1% Tween 20. Mock controls were treated with H₂O and 0.1% Tween 20 only. 23 days post inoculation, treated leaves were analyzed for infection symptoms in form of necrosis and pycnidia. Evaluation was performed by assigning categories for necrosis and pycnidia coverage to each leaf (categories: 0 = 0%, 1 = 1–20%, 2 = 21–40%, 3 = 41–60%, 4 = 61–80%, 5 = 81–100%).

Gene expression analysis

Expression of dnmt5 and rid in Zt09 and Zt10 as well as of dim2 in Zt10 was analyzed using previously generated in planta and in vitro RNA-seq data [35,41]. In planta data refers to the previously characterized morphological wheat infection stages: (A) infection establishment, (B) biotrophic growth, (C) lifestyle transition, (D) necrotrophic growth and reproduction [35]. In vitro data reflects growth of Zt09 in liquid YMS medium at 18°C and 200 rpm [41]. In brief, reads were quality filtered and trimmed as described in [35] and mapped to the respective Z. tritici genomes [37,71] using hisat2 v2.2.0 [90]. Relative abundance of transcripts for all Zt09 and Zt10 genes [46,71] was calculated in RPKM using Cuffdiff2 v2.1.1 [91].

Analysis of dinucleotide frequencies

TE sequences were extracted from the genome using bedtools getfasta [87]. k-mer frequencies in annotated TEs and masked genomes were determined using the software jellyfish (version 2.3.0) [92]. Correspondence analyses were carried out and visualized in R [93] using the packages "FactoMineR" (v2.3) and "factoextra" (v1.0.7) [94].

Mutation accumulation experiment and SNP analysis

A single colony derived directly from a plated dilution of frozen stock for Zt10 or IPO323 was resuspended in 1 mL YMS including 25% glycerol by 2 min vortexing on a VXR basic Vibrax at 2000 rpm, and 10–50 μL were re-plated onto a YMS agar plate. Forty replicates were produced. Cells were grown for 7 days at 18°C until a random colony (based on vicinity to a prefixed position on the plate) derived from a single cell was picked and transferred to a new plate as described above. The transfers were conducted for one year (52 times) before the DNA of a randomly chosen colony of each replicate was extracted and sequenced. Sequencing and library preparation were performed at the Max Planck Genome Center in Cologne, Germany (https://mpgc.mpipz.mpg.de/home/). Sequencing was performed on an Illumina HiSeq2500 machine obtaining paired-end 250-nt reads (S9 Table). Paired-end reads were quality filtered (Trimmomatic v0.38), mapped (bowtie2 v2.3.5) and SNPs were called using samtools (v1.7) mpileup (see S1 Text).