Additional file 10: of Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Figure S9. CNV of widely conserved genes between B. graminis formae speciales. Heatmap illustrating the copy number of genes with putatively widely conserved functions. Using the same pipeline as for the generation of Fig. 3a, all 34 genes with a PFAM annotation including the terms “tubulin” (highlighted in red) or “actin” (highlighted in green) and 49 genes coding for non-SP genes with conserved domains were used as a control dataset to estimate the error rate of the CNV calling pipeline. The heatmap depicts the color-coded copy number of these genes per individual genome of various B. graminis formae speciales (avenae, dactylis, dicocci, hordei, lolii, poae, secalis, triticale and tritici), each represented by one or more isolates as indicated on the right. The dendrogram on the left is based on the hierarchical clustering (Euclidean method) of the CNV values for every dataset. (PDF 466 kb

Additional file 5: of Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Figure S4. Comparative visualization of the genomic loci harboring AVR a1 and AVR a13 in the Bgh isolates DH14 and RACE1. (A) Organization of the genomic locus harboring the previously identified AVR a1 (orange arrows) and some of its flanking genes in DH14 and RACE1. (B) Organization of the genomic locus harboring the previously identified AVR a13 (green arrows) and some of its flanking genes in DH14 and RACE1. (PDF 1206 kb

Additional file 2: of Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Figure S1. Comparative alignment of the Bgh DH14 and RACE1 genome assemblies with a Bgh genetic map. The distribution and ordering of 80 single copy EST markers across 30 linkage groups of a previously published genetic map [22] is visualized in relation to the corresponding genomic locations of these markers in the DH14/RACE1 assemblies. Each box represents a specific genomic contig or linkage group (LG), respectively, and the numbers inside the boxes specify the marker positions on the corresponding contig (in bp) or linkage group (in cM). The corresponding marker identifiers are given next to the boxes. Dashed connector lines represent markers for which the genomic location and genetic map are consistent. Discrepancies between assembly and genetic map are indicated by solid connectors, with black lines representing markers whose location is consistent between assemblies but different from the genetic map, and colored lines representing markers with differences to the genetic map that are specific to either DH14 (dark pink) or RACE1 (blue). (PDF 4159 kb

Additional file 11: of Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Figure S10. Secretome orthology relations and core effectorome phylogeny. (A) Heatmap of SP orthologs found for the formae speciales genomes after ortholog clustering using OrthoFinder on the predicted proteomes of the isolates T1–20, S1459, LIB1609, DAC, 96224, LOL, AVE, POAE, DH14. Every column corresponds to one of the 805 Bgh DH14 predicted SPs, while color-coding depicts the number of orthologs in the corresponding orthogroup. Hierarchical clustering (Euclidean method) for the formae speciales and the SPs are given on the left and the top of the heatmap, respectively. (B) Maximum likelihood phylogeny tree of the 805 SPs. The tree was generated using IQ-TREE based on the mature peptide sequences of the Bgh DH14 SPs. Orange edge tips indicate the 190 core CSEPs which have orthologs in all formae speciales. The scale bar indicates the number of amino-acid substitutions per site. (PDF 2195 kb

Additional file 4: of Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Figure S3. Mitochondrial genomes of Bgh. (A) Map and corresponding annotation of the mitochondrial genome of Bgh isolate DH14 resulting from an RNAweasel and MFannot run. (B) Nucleotide sequence alignment between the DH14 (x-axis) and RACE1 (y-axis) mtDNA using NUCmer, indicating a putative partial duplication in RACE1. (PDF 224 kb

Additional file 6: of Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Figure S5. Variation in the mating type locus in the Bgh isolates DH14 and RACE1. Organization of the genomic loci containing the mating type genes (MAT-1-1-1, MAT-1-1-3 and MAT-1-2-1) and some of its flanking genes. As DH14 and RACE1 are of opposite mating types, the structure of the mating type locus differs between the two isolates. The genomic locus in RACE1, which is of the MAT-1-1 mating type, was assembled completely, while the respective locus in DH14 (MAT-1-2 mating type) is distributed on two scaffolds. (PDF 1139 kb

Additional file 9: of Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Figure S8. Distribution of SP and non-SP coding genes in Bgh DH14 scaffolds larger than 1 MB. (A) Density plots of SP coding genes (orange), non-SP coding genes (purple) and different types of TE elements (gray) in 50 kb sliding windows. Scaffolds depicted here were selected based on their size (> 1 MB) and represent ~ 87% of the total genomic sequence. (B) Number of SP coding genes per scaffold plotted against the respective total scaffold size, showing positive correlation (r = 0.88, p < 0.001). (PDF 4282 kb

Additional file 12: of Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Figure S11. Representatives of the genus Blumeria show less TE divergence than representatives of the genera Erishyphe and Golovinomyces. (A) The histograms indicate the frequency of a given sequence divergence for TE families of 10 B. graminis genomes. The genomes, which were assembled based on various sequencing platforms (PacBio or Illumina), were surveyed for their repeat content and repeat landscapes for each genome based on % nucleotide divergence to the consensus TE sequences were calculated out of the RepeatMasker output using Perl scripts. Sequence divergence (x-axis) is plotted against frequency (number of sequences; y-axis) for each of the genomes. (B) The histograms indicate the frequency of a given sequence divergence for TE families of 3 dicot-infecting powdery mildew species (Erysiphe pisi, E. necator and Golovinomyces orontii). The genomes, which were assembled based on various sequencing platforms (PacBio, ABI Solid or Illumina), were surveyed for their repeat content and repeat landscapes for each genome based on % nucleotide divergence to the consensus TE sequences were calculated out of the RepeatMasker output using Perl scripts. Sequence divergence (x-axis) is plotted against frequency (number of sequences; y-axis) for each of the genomes. (PDF 255 kb

Additional file 8: of Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen

Figure S7. Frequency of single-nucleotide polymorphisms (SNPs) between Bgh isolates. (A) Kernel density plot of the SNP frequencies per kb in 10 kb sliding windows, observed for the three Bgh isolates A6, K1 and RACE1 relative to the reference isolate DH14. The plot depicts Gaussian kernel density estimates calculated at a smoothing bandwidth of 0.12. (B) Average SNP frequencies for A6, K1 and RACE1 in 10 kb sliding windows of low and high SNP density as estimated by a two-component mixture model that was fitted to the observed SNP frequencies using the expectation-maximization algorithm. Error bars indicate the corresponding standard deviations estimated by the mixture model. (PDF 294 kb

Glutamate substitution analyses in the MLA10 CC domain identify the F99E autoactive mutation.

<p>(<b>A</b>) Analysis of cell death inducing activity of MLA10 CC mutant variants. MLA10 CC wild-type or mutant variants harboring indicated amino acid substitution were expressed in <i>N. benthamiana</i> leaves, and cell death induced by each protein was visualized by trypan blue staining at 42 hpi (upper panel). (<b>B</b>) Protein expression levels of each CC variant shown by Western blotting. Proteins were extracted from <i>N. benthamiana</i> leaves at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (<b>C</b>) Comparison of cell death inducing activity of MLA10 FL and the FL(F99E) variant. FL and FL(F99E) were expressed on the same <i>N. benthamiana</i> leaf, and the amount of cell death induced by each protein was shown by Trypan blue staining at 24 hpi (upper panel); protein expression levels of FL and FL(F99E) were shown by protein immunoblotting analysis using anti-HA antibody (bottom panel), protein extracts were obtained at ∼22 hpi. (<b>D</b>) Quantification of cell-death inducing activity of FL and FL(F99E). Upon expression of FL or FL(F99E) by Agro-infiltration in <i>N. benthamiana</i>, electrolyte leakage was measured each hour from 22 to 30 hpi, and then every two hours from 30 to 34 hpi; empty vector (EV) was included as a negative control. Error bars representing standard error (SE) were calculated from three replicates per time point and per construct. Similar experiments were repeated at least twice with similar results. Letters (a–c) represent significant differences [<i>p</i><0.05, Tukey's honest significant difference (HSD) test].</p