Genome-Wide Gene Expression Profiling of Fertilization Competent Mycelium in Opposite Mating Types in the Heterothallic Fungus Podospora anserina

are the major regulators of fertilization, and this study presents a genome-wide view of their target genes and analyzes their target gene regulation. strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. Interspecies comparisons of mating-type target genes revealed significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species.This study represents the first comprehensive genome-wide analysis of mating-type direct and indirect target genes in a heterothallic filamentous fungus. Mating-type transcription factors have many more target genes than are found in yeasts and exert a much greater diversity of regulatory actions on target genes, most of which are not directly related to mating

Systematic Deletion of Homeobox Genes in Podospora anserina Uncovers Their Roles in Shaping the Fruiting Body

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures

A RID-like putative cytosine methyltransferase homologue controls sexual development in the fungus Podospora anserina

International audienceDNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina

Fertility of the homeobox gene mutants.

<p>Repartition of fruiting bodies in homeobox mutants (top). Eight-centimeter M2 plates were inoculated in the center with mat+/mat− heterokaryotic cultures and incubated for seven days. Perithecia are visible as small black dots. Morphology of fruiting bodies in the Δ<i>pah</i> mutants (bottom). Images are from representative fruiting bodies from the plates in the top panel. For Δ<i>pah2</i>, arrows indicate perithecia with neck and arrowheads indicate perithecia without neck. WT, wild type.</p

Phylogenetic tree of fungi and the presence of croziers and clamps.

<p>The tree is based on the latest phylogenetic data including those of James et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#pone.0037488-James1" target="_blank">[3]</a>. In the Ascomycota, a smiling face indicates that croziers are present and a sad face indicates that they are absent. In the Basidiomycota, a smiling face indicates that clamps are present and a sad face indicates that they appear absent. No face is shown when we were not able to find data on the presence of croziers/clamps in the literature or that species from the group grow only as yeast.</p

Development of clamps and croziers shares several features.

<p>In both cases, division starts with the formation of a peg, lateral in Basidiomycota and apical in Ascomycota. Conjugate nuclear division then follows, resulting in one cell with two nuclei and two cells with one nucleus. Uninucleate cells undergo anastomosis to recreate the dikaryotic conditions. In Basidiomycota, the apical cell continues its vegetative growth and undergoes further cell division. In contrast, in most Ascomycota, the apical cell usually differentiates into a meiocyte and undergoes karyogamy and meiosis, although in a few species it may continue to divide as a dikaryon.</p

Evolution of HD transcription factors in Sordariomycetes.

<p>Top, alignment of the HD domains of the <i>P. anserina</i> HD transcription factors. The alignment was obtained with ClustalW2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#pone.0037488-Larkin1" target="_blank">[49]</a> and colored according to the ClustalX color scheme provided by Jalview <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#pone.0037488-Waterhouse1" target="_blank">[50]</a>. Numbers indicate the position in the protein of the first amino acids of the homeodomains. The bottom consensus line and the secondary structure are according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#pone.0037488-Brglin1" target="_blank">[9]</a>. Bottom, PhyML tree of the HD factors of <i>P. anserina</i>, <i>N. crassa</i> and <i>M. grisea</i>. Black dots indicate statistically supported branches (>80%, 200 bootstraps). Non-TALE HD in red, TALE HD in blue.</p

Crozier formation in the homeobox gene mutants.

<p>Perithecium centra were fixed, labeled with DAPI, and observed for the presence of croziers. Croziers were observed in all homeobox mutants, as shown here for Δ<i>pah1</i>. Some combinations of multiple mutants were also examined and found to differentiate croziers, as exemplified here by the Δ<i>pah2</i>Δ<i>pah3</i>Δ<i>pah4</i> triple mutant and the Δ<i>pah1</i>Δ<i>pah2</i>Δ<i>pah3</i>Δ<i>pah4</i>Δ<i>pah5</i>Δ<i>pah6</i> sextuple mutant.</p

Germination of WT and Δ<i>pah5</i> ascospores.

<p>Ascospores were incubated for 6 h at 27°C on germination medium and observed by light microscopy. <b>A.</b> WT ascospores from a WT <i>mat+</i> × WT <i>mat−</i> cross are melanized and germ tubes originate from a germination peg (*) located at the pole opposite to the primary appendage (arrowhead). <b>B.</b> Δ<i>pah5</i> ascospores from a trikaryotic culture (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#s2" target="_blank">Results</a>) are not fully melanized and germ tubes appear at both poles (arrow points at the germ tube originating from the primary appendage (*)). Numerous Δ<i>pah5</i> ascospores did not germinate even after incubation for several days.</p

Frequency of perithecia without neck in crosses homozygous for the Δ<i>pah</i> mutations.

a<p>Each cross was performed in duplicate on two separate Petri dishes inoculated with <i>mat+</i> and <i>mat−</i> strains. When the mycelia were confluent, sterile water was poured to disperse microconidia over the surface of the cultures and promote fertilization of female organs of the compatible mating-type. 5 to 9 independent counts were made seven days after fertilization on 150 to 400 perithecia formed on different sectors of the two dishes. Data from one*, two** or three*** assays were pooled. Δ<i>pah2</i> Δ<i>pah4</i>, Δ<i>pah2</i> Δ<i>pah6</i> and Δ<i>pah2</i> Δ<i>pah7</i> had the same % of perithecia without neck as Δ<i>pah2</i>. All the perithecia from Δ<i>pah1</i> Δ<i>pah5</i>, Δ<i>pah2</i> Δ<i>pah5</i>, Δ<i>pah3</i> Δ<i>pah5</i>, Δ<i>pah4</i> Δ<i>pah5</i> and Δ<i>pah6</i> Δ<i>pah5</i> had no neck. All the perithecia from the other double mutants had a beak.</p