Table 1.
HMG-box genes in P. anserina.
Figure 1.
Alignment of HMG domains from the 12 HMG-box proteins of P. anserina.
The alignment was performed using ClustalW2 [105] and colored according to the Clustal X color scheme provided by Jalview [106]. This color scheme is displayed in Table S1.
Figure 2.
Unrooted phylogram for the HMG-box superfamily.
Clustering of core amino acid sequences using maximum-likelihood and model LG+G [107]. Color labeling: MATα_HMG (A, green), MATA_HMG (B, yellow), SOX-TCF_HMG (C, orange), HMGB-UBF_HMG (D, H, blue), MAT1-1-3 in MATA_HMG (E, white) and STE11 in MATA_HMG (G, red). Other labels: Microsporidia MAT sex locus in HMGB-UBF_HMG (F, grey), Phycomyces blakesleeanus (Zygomycota) sexM (Phybl8) and sexP (Phybl9) (purple) and P. anserina proteins (red dots). LR-ELW values greater than 70% are shown. Abbreviations: Ailuropoda melanoleuca (Ailme); Ajellomyces capsulatus (Ajeca); Alternaria alternata (Altal); Alternaria brassicicola (Altbr); Anopheles gambiae (Anoga); Antonospora locustae (Antlo); Arabidopsis thaliana (Arath); Aspergillus fumigatus (Aspfu); Aspergillus nidulans (Aspni); Botryotinia fuckeliana (Botfu); Bipolaris sacchari (Bipsa); Caenorhabditis elegans (Caeel); Candida albicans (Canal); Cervus elaphus yarkandensis (Cerel); Ciona savignyi (Ciosa); Cochliobolus heterostrophus (Coche); Cochliobolus homomorphus (Cocho); Coprinopsis cinerea (Copci); Cryphonectria parasitica (Crypa); Culex quinquefasciatus (Culqu); Danio rerio (Danre); Dothistroma pini (Dotpi); Drosophila melanogaster (Drome); Enterocytozoon bieneusi (Entbi); Encephalitozoon cuniculi (Enccu); Fusarium acaciae-mearnsii (Fusac); Gibberella fujikuroi (Gibfu); Gibberella zeae (Gibze); Homo sapiens (Homsa); Lachancea thermotolerans (Lacth); Magnaporthe oryzae (Magor); Mycosphaerella graminicola (Mycgr); Podospora anserina (Podan); Neurospora crassa (Neucr); Penicillium marneffei (Penma); Phycomyces blakesleeanus (Phybl); Pneumocystis carinii (Pneca); Pyrenopeziza brassicae (Pyrbr); Pyrenophora teres (Pyrte); Rhynchosporium secalis (Rhyse); Saccharomyces cerevisiae (Sacce); Schizosaccharomyces japonicus (Schja); Schizosaccharomyces pombe (Schpo); Sordaria macrospora (Sorma); Stemphylium sarciniforme (Stesa); Strongylocentrotus purpuratus (Strpu); Takifugu rubripes (Takru); Ustilago maydis (Ustma); Verticillium dahliae (Verda); Xenopus laevis (Xenla); and Zygosaccharomyces rouxii (Zygro). Numbers after species names indicate α1 proteins (1), MATA_HMG (2), MAT1-1-3 (3), SOX (4), HMGB-UBF_HMG (5) and other HMG domains (6–9). When more than one domain was present for the same species, the suffix a, b or c was used. Units indicate the number of amino acid changes per position. Species codes and accession numbers grouped by evolutionary affinity are listed in Table S2.
Table 2.
Phenotypes of the P. anserina mutants with deleted HMG-box genes.
Figure 3.
Microscopic phenotypes of Δkef1 (ΔPahmg9) strain.
(A) Appressorium-like structures in the Δkef1 strain and in the wild type (WT). Development of appressorium-like structures was not affected in the Δkef1 strain. The pictures were taken every 1 µm as Z stacks, with “0” corresponding to hyphae growing onto the cellophane layer (see panel C). Arrowheads indicate needle-like hyphae that penetrated the cellophane layer; p: palm-like structures within the cellophane layer. (B) Comparison of hyphal fusion (anastomosis) in wild-type (WT) and Δkef1 strains. In the wild type, anastomoses were never observed in apical hyphae (1), while they were occasionally observed in the subapical area (2). In Δkef1 strain, anastomoses were profuse in apical (1) and in subapical (2) areas. Most notably, anastomosis occurred between the apex of hyphae at the leading edge and neighbouring apical hyphae (see arrows in Δkef1 in 1). (C) Schematic of appressorium-like development in P. anserina. a: appressorium-like structure including the palm-like structure and the needle-like hyphae; c: cellophane layer on which mycelium is growing. (D) Spermatia and their spermatogonia were present in the subapical area of the Δkef1 strain but were absent from the same area in the wild-type strain. Scale bar = 10 µm in all panels.
Figure 4.
Crosses of HMG-box gene deletion mutants.
(A) Analysis of male and female fertility of HMG-box gene deletion mutants in crosses with wild-type tester strains. When cultures were confluent, sterile water was poured and dispersed over the surface of the mycelium. As each strain produced spermatia (male cells) and protoperithecia (female organs) regardless of its mating type, reciprocal fertilization of the mutant and wild-type strains took place and indicated whether the mutant was fertile as a male (donor) or as a female (receptor). For ΔPahmg5 some perithecia differentiated at the contact zone where mutant and wild-type mycelia fuse. In the resulting heterokaryotic mycelium, wild-type nuclei complement the male and/or female sterility defect of the mutant. Those perithecia were fertile and expelled numerous asci allowing genetic analysis. (B) Analysis of perithecium distribution in homozygous crosses of HMG-box gene deletion mutants. Fragmented mycelium from mat+ and mat− strains with the same HMG-box deletion were deposited at the center of a Petri dish and incubated until perithecia formed. Typically, the wild-type strains differentiated perithecia within a ring-like area.
Figure 5.
Expression of HMG-box genes and mating-type target genes in strains with HMG-box gene deletion.
FCs from Table S3 to S7 were converted to a heat map using Matrix2png [108]. Squares for FCs with non-significant statistical values are in black. Squares for inapplicable values are in white. (A) Heat map in the mat+ strains. The FCs of HMG-box genes, FPR1 and selected FPR1 target genes are represented as indicated on the scale for the following strains: mat+ ΔPahmg6 (delPahmg6), mat+ Δmthmg1 (delmthmg1), mat+ Δkef1 (delkef1), mat+ ΔPahmg5 (delPahmg5), and mat+ ΔPahmg8 (delPahmg8). Gene number: MFP, Pa_2_2310; PRE2, Pa_4_1380; AOX, Pa_3_1710; PEPCK, Pa_4_3160. (B) Heat map in the mat− strains. The FCs of HMG-box genes, FMR1 and selected FMR1 target genes were represented as indicated on the scale in (A) for the following strains: mat− ΔPahmg6 (delPahmg6), mat− Δmthmg1 (delmthmg1), mat− Δkef1 (delkef1), mat− ΔPahmg5 (delPahmg5), and mat− ΔPahmg8 (delPahmg8). Gene number: MFM, Pa_1_8290; PRE1, Pa_7_9070.
Figure 6.
Genetic network of HMG-box genes that regulate mating in P. anserina.
Arrows with heads and blunt ends indicate activation and repression, respectively. The numbers next to the arrows indicate the average FC in gene expression between the wild-type and mutant strains.The relationship between mtHMG1 and PaHMG5 may be mediated by KEF1. Alternatively, mtHMG1 may by-pass KEF1 to repress PaHMG5 directly or indirectly. The consistency in the FCs suggest that PaHMG6 by-passes this cascade to activate PaHMG5 and PaHMG8 either directly or indirectly. The numbers next to the arrows connecting the mating-type genes (FMR1 and FPR1) and the downstream target genes are FCs that were obtained from [73].
Figure 7.
Conserved sequences found in the promoter region of HMG-box genes and mating-type target genes.
(A) Conserved sequences from a number of HMG-box genes and mating-type target genes identified using MEME program [78]. Position −1 is the first nucleotide upstream of the translation initiation codon. (B) Weblogo of the consensus sequence generated by MEME (assembled from sequences listed in A). (C) The P. anserina consensus sequence aligned with the binding site of HMG-box proteins: MATa-1 (N. crassa) [109], SpSte11 (S. pombe) [30], Prf2 (U. maydis) [38], [39] and Mat2 (C. neoformans) [41].
Figure 8.
Electrophoretic mobility shift assays with PaHMG5.
(A) Interaction of His tagged PaHMG5 (HMG5His) with probes corresponding to FMR1, FMR1 scrambled sequence (FMR1Sc), FPR1, PaHMG8 and PaHMG5 oligonucleotides. The probes are indicated below each panel. The interaction of HMG5His with probe was analyzed without competitor and in the presence of increasing amounts (given as fold molar excess below the triangles) of competitor (indicated above the triangles). (B) Interaction of the HMG5His protein with the MFM probe and reciprocal competition between FMR1 and MFM oligonucleotides. The probes are indicated below each panel. The competitors are indicated above the triangles. A control of the interaction of HMG5His with the FMR1 probe was performed as in A and included in the assay. HMG5His has a greater affinity for FMR1 than for MFM sequence, as indicated by the efficient exclusion of MFM probe by FMR1 competitor and the very inefficient exclusion of FMR1 probe by MFM competitor. (C) Interaction of the HMG5His protein with the MFP probe and reciprocal competition between FMR1 and MFP oligonucleotides. Legend as in B. HMG5His has a greater affinity for FMR1 than for MFP sequence, as indicated by the efficient exclusion of MFP probe by FMR1 competitor and the inefficient exclusion of FMR1 probe by MFP competitor. (D) Interaction of the HMG5His protein with the PRE1 probe and reciprocal competition between FMR1 and PRE1 oligonucleotides. Legend as in B. HMG5His has a greater affinity for FMR1 than for PRE1 oligonucleotides, as indicated by the efficient exclusion of PRE1 probe by FMR1 competitor and the inefficient exclusion of FMR1 probe by PRE1 competitor.