Fig 1.
Structural analysis of DNA methyltransferase proteins.
(A) Domain architecture of P. anserina putative DNA methyltransferase PaRid. The catalytic domains contain 10 conserved motifs (I—X) and a target recognition domain (TRD) located between the motifs VIII and IX. The amino acid length is indicated. (B) Domain architecture of DNA methyltransferase proteins (DMT). The functional domain analysis was performed using InterProScan and visualized using IBS. Mus musculus: MusmuDnmt1: NP_001300940.1, MusmuDnmt2: NP_034197.3, MusmuDnmt3A: NP_031898.1; Ascobolus immersus, AscimMasc1 (AAC49849.1), AscimMasc3; Aspergillus nidulans AspniDmtA: XP_664242.1, Neurospora crassa NeucrRid: AAM27408.1, NeucrDim2: XP_959891.1; Podospora anserina PaDim2: Pa_5_9100; PaRid: Pa_1_19440; Trichoderma reesei TrireDim2 XP_006964860.1; TrireRid: AEM66210.1. Cytosine-specific DNA methyltransferase domains: PF00145, PR00105, PS51679, PS00095, PS00094; protein-DNA interaction domains: bromo-associated homology (BAH) domain PS51038, Replication foci targeting sequence (RFTS) PF12047, Zinc finger motif (CXXC) PS51058, PWWP domain (PF00855). Because its sequence is too divergent, PaDnmt5 (Pa_4_2960) was not included.
Fig 2.
Phylogenetic analysis of mammal, plant and fungal DNA methyltransferases.
The maximum likelihood tree resolved five groups i) the Dnmt1/Met1/CMT group (orange), ii) the Dnmt2, group (black) iii) the Dnmt3/DRM group (red) iv) the fungal Dim2-like group (purple) and v) the fungal-specific Rid-like group (green), vi) the DNMT5 group (blue). Haemophilus aegyptius (HaeMTase: WP_006996493.1) Arabidopsis thaliana (ArathMet1: NP_199727.1; ArathCMT2: NP_193637.2; ArathCMT3: NP_177135.1; ArathDRM1: NP_197042.2; ArathDRM2: NP_196966.2), Ascobolus immersus (AscimMasc3: CE37440_11164; AscimMasc1: AAC49849.1; AscimDnmt5: RPA73956.1), Aspergillus nidulans (AspniDmtA: XP_664242.1, AspniDnmt5: XP_663680.1), Botrytis cinerea (BotciDim2: XP_024553164.1; BotciRid: XP_024550989.1, BotciDnmt5: XP_024550790.1), Cenococcum geophilum (CengeDim2: OCK96497.1; CengeRid: OCK89234.1), Coccidioides immitis (CocimDim2: XP_001247991.2; CocimRid: XP_001239116.2; CocimDnmt5: XP_001247253.2) Epichloe festucae (EpifeDim2: annotated in this study; EpifeRid: AGF87103.1), Fusarium graminearum (FusgrDim2: EYB34029.1; FusgrRid: XP_011320094.1) Pseudogymnoascus destructans (GeodeDim2: XP_024321957.1; GeodeRid: XP_024328520.1; GeodeDnmt5: XP_024320712.1) Magnaporthe grisea (MaggrDim2: XP_003718076.1; MaggrRid: XP_003720946.1) Mus musculus (MusmuDnmt1: NP_001300940.1; MusmuDnmt3A: NP_031898.1; MusmuDnmt3B: NP_001003961.2, MusmuDnmt2: NP_034197.3), Neurospora crassa (NeucrDim2: XP_959891.1; NeucrRid: AAM27408.1), Penicillium chrysogenum (PenchRid: XP_002563814.1; PenchDnmt5: XP_002561360.1), Podospora anserina (PaDim2: Pa_5_9100; PaRid: Pa_1_19440; PaDnmt5: Pa_4_2960), Pyronema confluens (Pyrco1dim2: PCON_02009m.01; Pyrco2dim2: PCON_01959m.01, PircoRid: PCON_06255m.01; PyrcoDnmt5: CCX08765.1), Schizosaccharomyces pombe (SchpoDnmt2: NP_595687.1), Thielavia terrestris (ThiteDim2: XP_003654318.1; ThiteRid: XP_003651414.1; ThiteDnmt5: XP_003650845.1), Trichoderma reesei (TrireDim2 XP_006964860.1; TrireRid: AEM66210.1), Trichophyton rubrum (TriruDim2: XP_003239082.1; TriruRid: XP_003239287.1; TriruDnmt5: XP_003236242.1), Tuber melanosporum (TubmeDim2: XP_002837027.1; TubmeRid: XP_002842459.1; TubmeDnmt5: XP_002837747.1).
Fig 3.
Evolution of the catalytic motifs IV and VI of the fungal Rid-like DNA methyltranferases.
A key catalytic step is the nucleophilic attack of the DNA methyltransferases on the sixth carbon of the target cytosine. This attack is made by the cysteine residue (red arrow) of the conserved PCQ triad (motif IV). This reaction is catalyzed by protonation of the N3 position of the cytosine by the glutamate residue of the conserved ENV triad (motif VI). In the Masc1/Rid-like group of enzymes, the ENV triad is replaced by either the EQT triad (e.g. N. crassa Rid, A. immersus Masc1, P. anserina PaRid, etc.) or the EET triad (e.g. B. cinerea Rid, P. destructans Rid, etc.). Arath: Arabidopsis thaliana, Musmu: Mus musculus, Cenge: Cenococcum geophilum, Maggr: Magnaporthe grisea, Trire: Trichoderma reesei, Fusgr: Fusarium graminearum, Neucr: Neurospora crassa, Pa: Podospora anserina, Geode: Pseudogymnoascus destructans, Botci: Botrytis cinerea, Cocim: Coccidioides immitis. Ascim: Ascobolus immersus, Tubme: Tuber melanosporum, Aspni: Aspergillus nidulans. See above for accession numbers of the corresponding proteins.
Fig 4.
PaRid is essential to complete fruiting body development and to produce ascospores.
(A) Homozygous crosses of wild type S strains (left panel) and of ΔPaRid strains (right panel) on M2 medium after 5 days at 27°C. Each dark dot is one fruiting body resulting from one event of fertilization. The homozygous ΔPaRid cross forms reduced-size fruiting bodies only (right panel). (B) Close up of fruiting bodies (perithecia) originating from either a wild-type genetic background (left panel) or a ΔPaRid genetic background (right panel). Scale bar: 250 μm. (C) After 4 days of growth at 27°C, the wild type fruiting bodies start to produce ascospores (left panel) while the mutant micro-perithecia are barren (right panel). Scale bar: 50 μm. (D) Fluorescence microscopy pictures of 48h-old fruiting body content from homozygous crosses of wild type S strains (left panel) and of ΔPaRid strains (right panel), performed on M2 medium at 27°C. The nuclei are visualized thanks to histone H1-GFP fusion protein. Croziers are readily formed inside the wild type perithecia (left panel, white arrows) while no crozier but large plurinucleate ascogonial cells only are seen inside the ΔPaRid perithecia (right panel, white arrow). Scale bar: 10 μm.
Table 1.
Complementation experiments.
Fig 5.
Expression and subcellular localization of PaRid during P. anserina life cycle.
PaRid-GFP expression was assayed from a ΔPaRid:AS4-PaRid-GFP-HA strain showing wild type phenotypes (PaRid-GFP). As a control, self-fluorescence was assayed from a wild-type strain (No GFP-tagged protein, PaRid+). No GFP signal can be observed in mycelium, however, a significant and specific GFP signal can be found in ascogonia (A, white arrows are pointing at ascogonia) and protoperithecia (B). Surprisingly, no GFP signal is observed in the croziers (C, white arrows are pointing at croziers) but a strong signal can be noticed in the mature ascospores (D, white arrows are pointing at the nuclei of one ascospore). Since self-fluorescence can be detected in the cytoplasm of ascospores, but not in the nuclei, the PaRid-GFP protein localization is nuclear. From left to right: bright-field (pol), DAPI staining, GFP channel and merge of the two latest. Scale bar: 1,5 μm (A and B), 5 μm (C and D).
Fig 6.
Transcriptomic analysis experimental design.
Schematic developmental time course from fertilization to ascospore maturation of wild-type crosses. Total RNA was extracted from wild-type perithecia at T24 and T30 (open circles) and from ΔPaRid micro-perithecia at T42 (solid circle); Dotted line: time frame during which the ΔPaRid developmental blockage might occur. Light microphotographs of the upper panel illustrate the various developmental steps indicated along the time course.
Fig 7.
Transcriptomic analysis of differentially expressed CDS in response to ΔPaRid developmental arrest.
(A) Functional categories in the down- and up-regulated CDS sets. Legend of pie charts corresponds to the FunCat categories, see Table 2 for details. Stars mark significantly enriched functional categories (p-value < 0.05). (B) Venn diagram of PaRid and FPR1 targets.
Table 2.
Main functional category analysis (FunCat).
Table 3.
Crosses used in transcriptome analysis.
Fig 8.
Schematic representation of PaRid & FPR1 developmental pathways during sexual development.
FPR1, a MATα-HMG transcription factor is essential to fertilization and development of fruiting bodies. This mating type protein can act either as an activator or as a repressor. This study established that PaRid shares part of the FPR1 positive regulatory circuit, which is at work after fertilization to build the fructification and to form the dikaryotic cells. As the methyltransferase activity is required for PaRid function, we hypothesized that PaRid might repress a repressor or alternatively might activate an activator of the FPR1 regulatory circuit [118]. Solid black arrow is indicative of activation. Dashed T-line is indicative of repression. Solid grey arrow represents the sexual development time line.