Figure 1.
A. Schematic phylogenetic tree showing the evolution of major fungal groups [128] and the position of H. werneckii, together with two other Mycosphaerella spp. with sequenced genomes. B. H. werneckii colonies on agar after two weeks at room temperature (above and middle) and microscopic image of the cell suspension at 1000× magnification (below).
Table 1.
Assembly and gene statistics for the Hortaea werneckii genome.
Figure 2.
Proteomes of Hortaea werneckii, Mycosphaerella graminicola and Saccharomyces cerevisiae.
A. Shared and unique proteins of three fungal species as determined by all-against-all blastp. Abbreviations used are. Hw: H. werneckii, Mg: M. graminicola and Sc: S. cerevisiae. B. Number of duplicated proteins. All predicted proteins were aligned to the genome by Exonerate and the number of possible alignment locations was counted for each protein. The proportion of proteins encoded by a certain number of gene copies are represented as columns of different heights (separately for each genome).
Figure 3.
Heterothallic Hortaea werneckii MAT loci.
A. Configuration of the MAT loci and adjacent genes in H. werneckii compared to Mycosphaerella graminicola. Arrows indicate the length and direction of transcription of the gene. Gene names are indicated above the gene model (black for exones, white for intrones). The models are drawn in scale (1 cm per 1000 nucleotides). B. Amino acid sequence alignment of HwMat1-1-1A (KC961394), HwMat1-1-1B (KC961395) and MgMat1-1-1 (XP_003847598.1) proteins. The MAT_Alpha1 conserved domain (PF04769) is indicated by a box, whereas the Alpha Box (PS51325, Prosite) is designated by dark grey.
Table 2.
Alkali metal cation transporter homologues in extremly halotolerant Hortaea werneckii.
Table 3.
Homologues of H+ -ATPases in extremly halotolerant Hortaea werneckii.
Figure 4.
Gene trees of various membrane transporters of inorganic ions from Hortaea werneckii (Hw), Mycosphaerella graminicola (Mg) and Saccharomyces cerevisiae (Sc).
The trees were rooted with homologous proteins from Cryptococcus neoformans and the root location is marked with an arrow. Putative gene duplications leading to the present diversity of these genes in H. werneckii are marked with different symbols: two triangles (duplications that happened before the separation of S. cerevisiae and H. werneckii ancestors), two half-circles (duplications after the separation of S. cerevisiae and H. werneckii ancestors, but before the separation of H. werneckii and M. graminicola), a combination of triangle and half circle (duplications before the separation of H. werneckii and M. graminicola but unclear regarding to separation from S. cerevisiae ancestor) and with circles on the bifurcation (recent duplications presumably resulting from a whole genome duplication). A. Plasma membrane transporters. B. Transporters located on internal membranes. The following S. cerevisiae transporters (Trk1 (YJL129C), Trk2 (YKR050W), Tok1 (YJL093C), Nha1 (YLR138W), Ena1 (YDR040C), Ena2 (YDR039C), Ena5 (YDR038C), Pho89 (YBR296C), Nhx1 (YDR456W), Kha1 (YJL094C), Vnx1 (YNL321W), Mrs7 (YPR125W)) and its H. werneckii (Table 2) and M. graminicola (Trk1: XP_003850012.1; Tok1: XP_003847595.1; Nha1: XP_003855439.1, XP_003856011.1 and XP_003855492.1; Ena1: XP_003852150.1, XP_003854801 and XP_003850456.1; Pho89: XP_003852378.1 and XP_003849212.1; Nhx1: XP_003850315.1; Kha1: XP_003852156.1; Vnx1:, XP_003854352.1, XP_003853630.1, XP_003849439.1 and XP_003852229.1; Mrs7: XP_003852324.1) homologues were included in the phylogenetic trees. Homologoues from C. neoformans (Trk1: XP_570017.1 and XP_569339.1; Tok1: XP_568987.1 and XP_568988.1; Nha1: XP_569560.1; Ena1: XP_572412.1, XP_568029.1 and XP_570160.1; Pho89: XP_568082.1; Nhx1: XP_570596.1; Kha1: XP_571501.1; Vnx1: XP_569752.1; Mrs7: XP_569566.1) were used as outgroups. The location of the root on the trees is marked by arrows.
Figure 5.
Gene tree of P-type ATPases from Hortaea werneckii, Ajellomyces dermatitidis, Leptosphaeria maculans, Mycosphaerella graminicola, Paracoccidioides brasiliensis, and Saccharomyces cerevisiae.
The specificity of each group of proteins is described in brackets immediately below the name of the group. Below this, numbers of proteins belonging to a given group in the proteomes of S. cerevisiae and H. werneckii (Sc/Hw) are shown, respectively.
Figure 6.
A. Plasma membrane ATPases (Pma). Gene phylogeny (above) of homologues from Hortaea werneckii (HwPma), Mycosphaerella graminicola (MgPma1: XP_003852209.1) and Saccharomyces cerevisiae (Pma1: YGL008C, Pma2: YPL036W), rooted by a homologue from Cryptococcus neoformans (XP_568571.1). Two half-circles mark a duplication after the separation of S. cerevisiae and H. werneckii ancestors, but before the separation of H. werneckii and M. graminicola, black circles on the bifurcation mark recent duplications presumably resulting from a whole genome duplication. Transcription profiles (below) of plasma membrane H+-ATPases of H. werneckii homologues at different concentrations of NaCl (w/v). Quantitative reverse transcription PCR (qRT-PCR) was performed with RNA isolated from cells grown in YNB medium, supplemented with 0, 5, 10, 17, and 25% NaCl (w/v). Quantification cycle (Cq) values for our genes of interest were normalised to the quantification cycle of 28S rRNA fragment (reference gene). The difference in Cq values (relative mRNA level values) between the target gene and the reference gene was calculated, and these values of the different samples were compared directly. Data are means of relative mRNA level values obtained by two qRT-PCR experiments performed with biological triplicates. B. The subunit A of the vacuolar ATPases (Vma1). Gene phylogeny (above) of homologues from Hortaea werneckii (HwVma), Mycosphaerella graminicola (MgVma1: XP_003850333.1) and Saccharomyces cerevisiae (Vma1: YDL185W), rooted by a homologue from Cryptococcus neoformans (XP_570895.1). Black circles on the bifurcation marks a recent duplication presumably resulting from a whole genome duplication. Transcription profiles (below) of vacuolar H+-ATPases of H. werneckii homologues at different concentrations of NaCl (w/v). Quantitative reverse transcription PCR (qRT-PCR) experiment and the analysis of the data was performed as described above.