The crucial role of the Pls1 tetraspanin during ascospore germination in Podospora anserina provides an example of the convergent evolution of morphogenetic processes in fungal plant pathogens and saprobes

Abstract

Pls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina, a saprobic fungus lacking appressorium, contains PaPls1, a gene orthologous to known PLS1 genes. Inactivation of PaPls1 demonstrates that this gene is specifically required for the germination of ascospores in P. anserina. These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1, which fully complements a DeltaPaPls1 ascospore germination defect, has no role in the germination of Magnaporthe grisea nonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserina mutants with mutation of PaNox2, which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the DeltaPaPls1 mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserina melanized ascospores. The analysis of the distribution of PLS1 and NOX2 genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserina ascospores and the formation of the M. grisea appressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.

FIG. 1.

Alignment and phylogenetic tree of Sordariomycetes and Leotiomycetes Pls1 tetraspanins. (A) Pls1 proteins from Sordariomycetes and Leotiomycetes display numerous conserved motifs in transmembrane domains (TM1 to -4 [underlined in black]), in ECL2 including a cysteine pattern (underlined in gray), and in the C-terminus tail. Protein sequences were aligned using ClustalX 1.8. Conserved amino acids are boxed in black (identical) and gray (similar). (B) PaPls1 clusters with CgPls1 and NcPls1, as expected, since the corresponding fungal species are closely related. The phylogenetic tree was constructed using the previous alignment (A) and a maximum likelihood method (PHYML) with BcPls1 and ScPls1 as outgroups. Bootstrap values are expressed as percentage of 100 replicates. Abbreviations for Sordariomycetes: Pls1_Pa, Podospora anserina Pa_1_19270; Pls1_Cg, Chaetomium globosum CHGG_06472.1; Pls1_Nc, Neurospora crassa AJ504996; Pls1_Cl, Colletotrichum lindemuthianum AJ504995; Pls1_Mg, Magnaporthe grisea AX058239; Pls1_Fg, Fusarium graminearum FG08695.1; Pls1_Tr, Trichoderma reesei jgi Trire2 4514 fgenesh1_pm.C_scaffold_13000033. Abbreviations for Leotiomycetes: Pls1_Bc, Botrytis cinerea AJ504994; Pls1_Ss, Sclerotinia sclerotiorum SS1G_05586.1.

FIG. 2.

Time course of the germination of a wild-type P. anserina ascospore on inducing medium. P. anserina ascospores are bicellular, with one large heavily melanized cell and a small hyaline one, the primary appendage (arrowheads). The germination starts with the extrusion of a peg (black arrow) at the pole opposite to the primary appendage, where the germ pore is located (3). The white arrow points to a newly formed hypha with polarized growth issued from the spherical germination peg. This hypha appears 20 min after the germination peg.

FIG. 3.

Lack of P. anserina ascospore germination in ΔPaPls1 and ΔPaNox2 mutants. Germination of melanized (main panels) and unmelanized (inserts) ascospores on inducing medium. Ascospores from the main panels originate from crosses between wild type and wild type, ΔPaPls1 and ΔPaPls1, and ΔPaNox2 and ΔPaNox2 strains of opposite mating types. Ascospores from the inserts originate from crosses between the wild type and pks1-193 (WT), ΔPaPls1 and pks1-193 ΔPaPls1 (ΔPaPls1), and ΔPaNox2 and ΔPaNox2 pks1-193 (ΔPaNox2) strains. Arrows point to germination pegs. In the main panels, melanized ascospores from ΔPaPls1 and ΔPaNox2 mutants are unable to germinate, while wild-type ascospores do germinate. In the inserts, ΔPaPls1 and ΔPaNox2 melanized ascospores did not germinate, while several ΔPaPls1 and ΔPaNox2 unmelanized ascospores did germinate, demonstrating that the inhibition of melanin biosynthesis suppresses ΔPaPls1 and ΔPaNox2 defects. In the wild-type × pks1-193 cross (WT insert), only the nonmelanized ascospores have germinated when the picture was taken, indicating that ascospores lacking melanin germinate before pigmented ones.

FIG. 4.

Ontogeny of ascospore and appressorium in P. anserina and M. grisea. (A) Formation of ascospores in M. grisea results in four-cell hyaline ascospores that germinate spontaneously (55). Ascospores were observed 16 h after deposition onto water agar (4.5% agar) at 26°C. The picture shows the germination of a wild-type ascospore that occurred at two opposite tips of the spore. (B) Formation of ascospore in P. anserina results in two-celled ascospores (3). One cell undergoes enlargement and melanization, while the other undergoes programmed cell death to form the primary appendage (*). Ascospores were observed 4 h after deposition onto germination medium at 27°C. Germination proceeds only on a special medium rich in ammonium acetate through the germ pore (arrow), which is located opposite of the dead cell. The picture shows the germination of a typical wild-type ascospore with its germination peg. (C) Appressoria are formed at the tip of the germ tube issued from the germination of a three-cell spore produced by asexual reproduction on contact with an hydrophobic hard surface (8). The single-cell appressorium strongly adheres to the plant surface and penetrates its host through the formation of a penetration peg at a basal pore as depicted in the picture.