PMC

High diversity in melanin levels is exhibited among strains from four worldwide populations. Gray values of 37 different Z. tritici strains from four different field populations across the world. The colors of the bars indicate the population to which the strains belong. Means and standard errors of the gray values were based on colonies grown for 7 days. Some examples of Z. tritici strains illustrating the diversity in melanin accumulation are shown in the lower panel. The experiment was performed three times with similar results

The transposable element insertion upstream of Zmr1 influences the expression of adjacent genes. a Schematic representation of the Zmr1 locus in the wild-type 3D7 and the insertion of a hygromycin resistance cassette (Hyg.R) in 3D7Δzmr1 and in the ectopic controls. b Growth of 3D7, 3D7Δzmr1, and the ectopic controls in yeast malt sucrose (YMS) plates with and without hygromycin (100 μg/ml). c Schematic representation of the Zmr1 locus in the wild-type 3D1 and the insertion of a hygromycin resistance cassette (Hyg.R) in 3D1Δzmr1 and in the ectopic controls. d Growth of 3D1Δzmr1 was reduced compared to growth of the ectopic controls in the presence of hygromycin (100 μg/ml). Growth is normal for all lines in the absence of hygromycin. The experiment was performed three times with similar results

Zmr1 expression is higher in the darker strain (3D7) compared to 3D1. Zmr1 mean expression and standard deviation in 3D1 and 3D7 at 7 and 9 days post inoculation (dpi) relative to the expression of Zmr1 in 3D1 at 7 dpi. Colonies grown on at least three different YMS plates were pooled for RNA extraction. The experiment was performed three times with similar results. Asterisks represent significant differences between 3D1 and 3D7 (p value ≤ 0.05, Kruskal-Wallis test). A representative picture of each strain is shown at 7 and 9 dpi

Sequence variation in the Zmr1 promoter contributes to differences in melanin accumulation. Gray values for 3D1, 3D7, the Zmr1 knockout in 3D7 (3D7Δzmr1), three in locus complementation transformants (3D7Δzmr1 + Zmr1_3D7 #4, #11, #23) and three in locus allele swap mutants of Zmr1 (3D7Δzmr1 + Zmr1_3D1 #12, #14, #72), all grown for 7 days. Asterisks (*) and plus (+) indicate significant differences in gray values of each strain with respect to the gray value of 3D7Δzmr1 and 3D7, respectively (p value ≤ 0.05, Kruskal-Wallis). At least 20 colonies (replicates) grown on three different plates were evaluated. The experiment was performed twice with similar results

Melanin accumulation is associated with reduced growth and resistance to SDHI fungicides. a, b Colony radius over time of melanin-deficient mutants in 3D1 (a) and 3D7 (b) backgrounds. The experiment was performed three times with similar results. c Melanin protects Z. tritici against the SDHI fungicide bixafen. Percentage decrease in growth of the wild-type 3D7 and the 3D7Δzmr1 knockout in the presence and absence of the fungicide at each time point (8–12 dpi). Mean and standard error of differential radial size of colonies grown on three independent plates are presented. The experiment was performed twice with similar results. Asterisks (*) indicate statistical differences between wild-type and knockout at each time point (p value ≤ 0.05, Kruskal-Wallis). ns non-significant

Differences between 3D1 and 3D7 in Zmr1 regulatory sequences. a Alignment of the nucleotide sequences 1862 bp upstream of the coding sequence of Zmr1 in 3D1 and 3D7. Vertical pink bars indicate the 12 SNPs in the promoter region. b Synteny plot of the QTL between 3D1 and 3D7 showing the insertion of an island of transposable elements 1.8 kb upstream of the start codon of Zmr1 in the lighter strain 3D1. Brown lines indicate collinear sequences. The positions of genes and transposable elements are shown using purple arrows and blue bars, respectively. Vertical yellow lines indicate simple repeats. The different shades of blue represent different classes of transposable elements that were classified according to the three-letter code described in Wicker et al. []. The first letter indicates the class (R = RNA class and D = DNA class), the second letter indicates the order (L = LTR, I = Line, T= TIR), and the third letter indicates the superfamily (G = Gypsy, I = I, X = unknown)

The transposable element insertion in the lighter strain downregulates Zmr1 expression. a Deletion of the transposable element island leads to significantly more melanin accumulation in 3D1 at 7 days post inoculation (dpi). Distribution of gray values for at least 35 colonies at 7 dpi for 3D1, 3D7, the transposable element deletion mutants in the 3D1 background (3D1ΔTE #93, #131 and #239), and the ectopic controls (#3 and #6). Asterisks indicate significant differences in gray values with respect to the wild-type 3D1 (p value ≤ 0.05, Kruskal-Wallis). The experiment was performed three times with 3D1ΔTE #93 and twice with #131 and #239 and we obtained similar results. b Zmr1 expression levels in the transposable element knockouts (#93, #131, #239) are significantly higher than those in the wild-type 3D1 at 7 dpi. Zmr1 expression values are relative to the expression of Zmr1 in 3D1. Means and standard deviations of three technical replicates are shown. Asterisks (*) represent statistical differences with the wild-type (p value ≤ 0.05, Kruskal-Wallis test). The experiment was performed twice and we obtained similar results

Variability in melanization levels between 3D1 and 3D7 maps to the Pks1 cluster. a The Swiss strain 3D1 is less melanized than 3D7. Melanization levels of 3D1 and 3D7 at 7 to 12 days. Bars represent standard errors of the mean gray value based on at least 60 colonies. Asterisks indicate significant differences according to Kruskal-Wallis test (p value ≤ 0.05; ns non-significant). Representative pictures of both strains are shown below the bar plot for all the time points. The experiment was performed three times with similar results. Gray value scale (0 = black, 255 = white) is shown on the left. b Genes in the 95% confidence interval of the QTL mapped to the genomes of the reference strain IPO323 and of the parental Swiss strain 3D7. The shift in the position of the QTL, genes with and without non-synonymous mutations, and the positions of the QTL peaking markers are indicated

Transposable element insertions regulate Zmr1 expression and melanin accumulation in Z. tritici populations. a Schematic representation of the location and classification of the transposable element insertions upstream of Zmr1 in different Z. tritici strains from a worldwide collection. The transposable elements were classified according to the three-letter code described in Wicker et al. (2007) []: The first letter indicates the class (R = RNA class and D = DNA class); the second letter indicates the order (I = Line, T= TIR, X = unknown); and the third letter indicates the superfamily (L = L1, M = Mutator, T = Tc1-Mariner, X = unknown). b Transposable element insertions upstream of Zmr1 significantly contribute to a reduction in melanin accumulation, according to Kruskal-Wallis (p value = 0.0008, indicated with asterisks). Gray value distributions of Z. tritici strains with and without transposable element insertions upstream of Zmr1. The experiment was performed three times and we obtained similar results. c Transposable element insertions upstream of Zmr1 negatively affect Zmr1 expression (Kruskal-Wallis, p value = 0.035, indicated with asterisks). Distribution of the mean expression of Zmr1 (relative to 3D1 at 7 days post inoculation) in each Z. tritici strain with and without transposable element insertions upstream of Zmr1. The experiment was performed twice with similar results