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 ⌬PaPls1 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 ⌬PaPls1 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.Convergent evolution of trophic strategies and morphogenetic processes is a hallmark of fungi (27). For example, many fungi belonging to different fungal clades are phytopathogenic, whereas their close relatives are saprobic. These plant parasitic fungi may penetrate into their host plants using a specialized structure called the appressorium. Appressoria are attached to the surface of plants and redirect fungal growth into the host tissues underneath through the formation of a penetration peg. Due to their importance in pathogenicity, appressoria are studied in fungi such as Magnaporthe grisea, a devastating pathogen of rice (25, 54), Botrytis cinerea, a wide-host-range plant pathogen (19), and Colletotrichum lindemuthianum, an important pathogen of beans (13). These three fungi belong to two different clades of ascomycetes. M. grisea and C. lindemuthianum are in the Sordariomycetes and B. cinerea in Leotiomycetes. A significant number of species related to these three fungi are saprobic. Which of the parasitic or saprobic lifestyles displayed by these organisms arise by convergent evolution is thus crucial to understand their evolution. An answer to this question is to establish whether appressoria are homoplasious or homologous in Sordariomycetes and Leotiomycetes and whether appressorium-specific ...

Maintaining Two Mating Types: Structure of the Mating Type Locus and Its Role in Heterokaryosis inPodospora anserina

“…To check that we correctly identified the borders of the recombination-less region, we genetically mapped several markers with respect to the mating type (Figure 1). The markers were obtained by gene replacements or classical genetics in diverse projects prior to this study (Silar and Picard 1994;Silar et al 1997;Graïa et al 2000;Contamine et al 2004;Bonnet et al 2006;Lambou et al 2008;J. Ait-Benkhali, E. Coppin, and R. Debuchy, unpublished data).…”

New Insights Into the Roles of NADPH Oxidases in Sexual Development and Ascospore Germination in Sordaria macrospora

“…We found that NOX2 is required for germination of melanized ascospores only, since melanin-deficient ascospores from Δnox2 are able to germinate. Lambou et al (2008) suggested for P. anserina that the weakened cell wall of pigment-deficient ascospores and the absence of melanin either triggered the germination process or prevented its inhibition. However, the ROS scavenging capacity of melanin (Riley 1997) might also play a role in this process.…”

“…Another candidate NOX regulatory protein is tetraspanin Pls1. Strains from different ascomycetes, lacking the corresponding gene, have phenotypes similar to nox2 deletion strains (Lambou et al 2008;Ryder et al 2013;Siegmund et al 2013). However, very little is known about the upstream and downstream components of the NOX signaling pathways in fungi.…”

A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway inPodospora anserina

“…The DPaMpk1, IDC 404 PaMKK1, and IDC 118 PaASK1 mutants have been previously described and differ from wild type by a single mutation (Kicka and Silar 2004;Kicka et al 2006) as do the pks1-193 mutants (Coppin and Silar 2007). Construction of the Dmus51::su8-1 strain lacking the mus-51 subunit of the complex involved in end joining of broken DNA fragments was described previously (Lambou et al 2008). DNA integration in this strain proceeds almost exclusively by homologous recombination.…”

“…A XhoI site in M3K2DF and an EcoRV one in M3K2DR allowed the release from the pGEMT vector of the corresponding 39 noncoding fragment, which was then cloned at the XhoI/ EcoRV sites into the plasmid harboring the PaMkk3 59 noncoding fragment, generating the pDPaMkk3 plasmid. Transformation of the P. anserina Dmus51::su8-1, pks1-193 strain (Lambou et al 2008) was performed using pDPaMkk3 plasmid previously linearized at the unique EcoRV site to generate homologous recombination ends. Three hygromycine B-resistant transformants were selected for further analyses.…”