Alex Mercier

Colin Clairet

Robert Debuchy

David Morais

Philippe Silar

Dr Sylvain Brun
email: sylvain.brun@univ-paris-diderot.fr

The mitochondrial translocase of the inner membrane PaTim54 is involved in defense response and longevity in Podospora anserina

Keywords: Tim54, Hyphal Interference, aging, mitochondria, Podospora anserina, fungi

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Fungi are very successful microorganisms capable of colonizing virtually any ecological niche where they must constantly cope with competitors such as other fungi, bacteria, nematodes [START_REF] Boddy | Interspecific combative interactions between wood-decaying basidiomycetes[END_REF][START_REF] Boddy | Fungal Ecology: Principles and Mechanisms of Colonization and Competition by Saprotrophic Fungi[END_REF][START_REF] Crowther | Functional and ecological consequences of saprotrophic fungus-grazer interactions[END_REF]. Fungi are often engaged in multiple kinds of biotic interactions ranging from symbiotic to antagonistic [START_REF] Devay | Mutual relationships in fungi[END_REF]. In particular, saprotrophic fungi that compete for nutrients have developed strategies to avoid spreading of competitors. Antibiosis, the most famous and the most studied antagonistic interaction is viewed as a long-distance process that mainly relies on the capacity of fungi to constitutively produce and secrete toxic secondary metabolites [START_REF] Macheleidt | Regulation and Role of Fungal Secondary Metabolites[END_REF]. Hyphal interference (HI) and mycoparasitism are on the contrary responses engaged after direct contact with challengers. Triggering such antagonistic interactions implies that fungi must be endowed with a non-self-recognition system to adapt the nature of the interaction with the competitor [START_REF] Silar | Hyphal Interference: Self Versus Non-self Fungal Recognition and Hyphal Death[END_REF][START_REF] Uehling | Do fungi have an innate immune response? An NLR-based comparison to plant and animal immune systems[END_REF]. Non-self-recognition has been studied in Vegetative Incompatibility (VI) observed in both ascomycete model systems Neurospora crassa and Podospora anserina but this phenomenon involves competitors from the same species [START_REF] Glass | The Genetics of Hyphal Fusion and Vegetative Incompatibility in Filamentous Ascomycete Fungi[END_REF][START_REF] Saupe | Vegetative incompatibility in filamentous fungi: Podospora and Neurospora provide some clues[END_REF]. On the contrary, HI (and mycoparasitism) takes place between competitors from different species. HI was first investigated by Ikediugwu and Websters who demonstrated that the basidiomycete Coprinus heptemerus kills hyphal cells of several ascomycetes competitors at the confrontation between thalli [START_REF] Ikediugwu | Ultrastructure of hyphal interference between Coprinus heptemerus and Ascobolus crenulatus[END_REF]Ikediugwu et al., 1970;Ikediugwu and Webster, 1970). Although in both VI and HI, the eventual outcome is the death of fungal cells, the major discrepancy between VI and HI is the oxidative burst observed in HI only [START_REF] Silar | Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant[END_REF]. Indeed, when P. anserina is challenged by Penicillium chrysogenum, hydrogen peroxide accumulates on the thallus of P. anserina at the confrontation and it has been shown that cells of P. chrysogenum in contact with those of P. anserina are killed. This latter feature of HI resembles the oxidative burst taking place during innate immune response that kills or limit the spreading of invading pathogens in both plants and animals [START_REF] Iles | Macrophage signaling and respiratory burst[END_REF][START_REF] Liu | Regulation of plant reactive oxygen species (ROS) in stress responses: learning from AtRBOHD[END_REF][START_REF] Marino | A burst of plant NADPH oxidases[END_REF]. This analogy with the innate immune response of higher Eukaryotes was further strengthened when it was shown in P. anserina that HI requires the NADPH oxidase (Nox) PaNox1, the homologue of mammalian NOX2/gp91 phox and of plant Rboh O2 --producing enzymes [START_REF] Silar | Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant[END_REF]. Indeed, when the PaNox1 -mutant is challenged with P. chrysogenum, the accumulation of hydrogen peroxide on the mutant thallus on the one hand and cell death in P. chrysogenum on the other hand are decreased at the confrontation of both mycelia. Noteworthy, three Nox isoforms are found in fungi, Nox1/A, Nox2/B and Nox3/C. While the role of Nox3/C remains unknown, Nox1/A and/or Nox2/B regulate an astonishing number of processes such as cellulose degradation, anastomosis (cell fusion), sexual reproduction, ascospore germination, cytoskeleton remodeling, symbiosis and pathogeny [START_REF] Aguirre | Reactive oxygen species and development in microbial eukaryotes[END_REF][START_REF] Brun | Functions and regulation of the Nox family in the filamentous fungus Podospora anserina : a new role in cellulose degradation[END_REF][START_REF] Scott | Conservation of fungal and animal nicotinamide adenine dinucleotide phosphate oxidase complexes: Conservation of animal and fungal Nox[END_REF][START_REF] Scott | Role of reactive oxygen species in fungal cellular differentiations[END_REF][START_REF] Tudzynski | Reactive oxygen species generation in fungal development and pathogenesis[END_REF]. However, only PaNox1 regulates HI [START_REF] Brun | Functions and regulation of the Nox family in the filamentous fungus Podospora anserina : a new role in cellulose degradation[END_REF][START_REF] Silar | Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant[END_REF].

In P. anserina, IDC 343 , the mutant of PaNox1 was isolated in a screen designed to identify mutants of Crippled Growth (CG), an epigenetically triggered mycelial degeneration observed when P. anserina is grown on specific media [START_REF] Haedens | Genetic control of an epigenetic cell degeneration syndrome in Podospora anserina[END_REF][START_REF] Silar | Propagation of a Novel Cytoplasmic, Infectious and Deleterious Determinant Is Controlled by Translational Accuracy in Podospora anserina[END_REF]. Several Impaired Development of CG (IDC) mutants proved to be pleiotropic mutants defective in processes such as HI. In particular, mutants of the PaNox1 complex or of the Cell Wall Integrity pathway (CWI) PaMpk1 MAP Kinase pathway, are IDC mutants that exert similar HI defects as PaNox1 [START_REF] Brun | Functions and regulation of the Nox family in the filamentous fungus Podospora anserina : a new role in cellulose degradation[END_REF][START_REF] Lacaze | Identification of NoxD/Pro41 as the homologue of the p22 phox NADPH oxidase subunit in fungi: NoxD/Pro41: the fungal p22 phox homologue[END_REF][START_REF] Silar | Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant[END_REF]. Not only IDC mutants have been isolated in the original screens for CG mutants:

Promote Development of CG (PDC) mutants were also isolated and tested for HI. Amongst these IDC and PDC mutants, the PDC 2218 mutant showed unusual phenotype for HI. When challenged with P. chrysogenum, the PDC 2218 mutant produces a massive oxidative burst at the confrontation. However, this increased production of hydrogen peroxide by the PDC 2218 mutant is not correlated to increased cell death in P. chrysogenum [START_REF] Silar | Hyphal Interference: Self Versus Non-self Fungal Recognition and Hyphal Death[END_REF]. In this mutant, recognition of P. chrysogenum likely occurs but the triggered response appears abnormal. Especially, the oxidative burst and cell death in the challenger are uncoupled, which strengthen the previous assumption that hydrogen peroxide has a signaling role rather than a toxic one [START_REF] Brun | Functions and regulation of the Nox family in the filamentous fungus Podospora anserina : a new role in cellulose degradation[END_REF][START_REF] Silar | Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant[END_REF]. With the aim to get insights into the link between the oxidative burst in P. anserina and HI-induced cell death in the challenger, the identification of the gene affected in PDC 2218 was undertaken. We first proceeded to positional cloning of the mutation and we identified a deletion spanning 1560 bp in the mutant genome. Even though the deletion disrupts the 5' part of the PaNip30 putative CDS as well as part of the 3' UTR (UnTranslated Region) of the neighboring PaCdk5 putative gene, our investigations show that none of these genes are important for HI. Indeed, by a combination of different approaches: functional complementation, gene deletion, genetic analyses and gene expression data, we show here that the gene important for creating the HI phenotype in PDC 2218 is PaTim54, the gene encoding the homologue of the budding yeast mitochondria inner membrane carrier Tim54p [START_REF] Hwang | Tim54p connects inner membrane assembly and proteolytic pathways in the mitochondrion[END_REF][START_REF] Kerscher | The Tim54p-Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane[END_REF][START_REF] Kurz | Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway[END_REF]. Surprisingly, this gene is located more than 1000 bp away from the deletion. We show that PaTim54 is essential in P. anserina and that the phenotypes displayed by the hypomorphic PDC 2218 mutant (renamed PaTim54 2218 ) are the consequence of a drastic reduction in the expression of PaTim54.

Contrary to many filamentous fungi, P. anserina has a limited vegetative growth phase that terminates by a the senescence of the cells at the thallus apex (Rizet, 1953a). For decades, this fungus has been a successful model system in studying aging and especially the effects of mitochondrial disorders in this degenerative process [START_REF] Lorin | Mitochondrial metabolism and aging in the filamentous fungus Podospora anserina[END_REF][START_REF] Osiewacz | Mitochondrial pathways governing stress resistance, life, and death in the fungal aging model Podospora anserina[END_REF]. In this paper, we show that the PaTim54 2218 mutation increases longevity and alters the respiratory chain, mitochondrial DNA (mtDNA) nucleoids as well as mitochondria morphology.

Materials and methods

Strains, culture conditions and DNA preparations

All the strains used in this study (Table 1) derived from the "S" (big S) wild-type strain that was used for sequencing the P. anserina genome [START_REF] Espagne | The genome sequence of the model ascomycete fungus Podospora anserina[END_REF]. The genome sequence and EST derived from the S strain are available at http://podospora.i2bc.paris-saclay.fr/. The polymorphic P. comata strain T was used for positional cloning of the PDC 2218 mutation [START_REF] Boucher | Species Delimitation in the Podospora anserina/ p. pauciseta/p. comata Species Complex (Sordariales)[END_REF]. The PDC 2218 (PaTim54 2218 ) and the PaNox1 -(IDC 343 ) strains were first described in [START_REF] Haedens | Genetic control of an epigenetic cell degeneration syndrome in Podospora anserina[END_REF]; the ΔPaMpk1 in [START_REF] Kicka | A mitotically inheritable unit containing a MAP kinase module[END_REF] and the mito-GFP strain in [START_REF] Sellem | A mutation in the gene encoding cytochrome c1 leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina[END_REF]. All the deletions by homologous recombination have been performed in strains inactivated for Pamus51, the gene encoding the helicase of the NHEJ repair system, enhancing homologous recombination in these strains [START_REF] El-Khoury | Gene deletion and allelic replacement in the filamentous fungus Podospora anserina[END_REF]. Standard culture conditions, media and genetic methods for P. anserina are reported in http://podospora.i2bc.paris-saclay.fr/; the M2 standard medium is a medium in which carbon is supplied as dextrin, and nitrogen as urea. The methods used for nucleic acid extraction and manipulation have been described [START_REF] Lecellier | Rapid methods for nucleic acids extraction from Petri dish-grown mycelia[END_REF]. Transformation of P. anserina protoplasts was carried out as described previously [START_REF] Debuchy | Cloning of opal suppressor tRNA genes of a filamentous fungus reveals two tRNASerUGA genes with unexpected structural differences[END_REF].

Deletion of PaCdk5

The PaCdk5 (Pa_6__9000) predicted CDS was deleted by a split-marker strategy [START_REF] Lalucque | A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina[END_REF]. To construct the deletion cassette of the entire PaCdk5 coding sequence, a hygromycin B resistance (hygR) marker was fused by PCR with the upstream or the downstream sequence of the PaCdk5 gene (Fig. S1). 1063 bp upstream and 1009 bp downstream of PaCdk5 were amplified from the wild-type genomic DNA with primers 9000_1 and 9000_2 for the upstream region and 9000_10 and 9000_11 for the downstream region (Table S1). Primers 9000_2 and 9000_10 contained additional bases allowing fusion with the hygR cassette. The hygR cassette was amplified using primers 9000_HYGF/9000_HYGR with pBC-hyg plasmid as template [START_REF] Silar | Two new easy to use vectors for transformations[END_REF]. In a second step, two amplification reactions were performed using primers 9000_1/HY and YG/9000_11 to obtain the fusion of two PCR products, the upstream (5') region/hygR and hygR/downstream (3') region. The mixture of these two PCR products was used to transform a ∆mus51::phleoR strain (El- [START_REF] El-Khoury | Gene deletion and allelic replacement in the filamentous fungus Podospora anserina[END_REF] All the transformants showed a similar mutant phenotype (Figure 1). For twelve of them, integration of the deletion K7 was checked by PCR using primers 9000_5/HygL for the 5' flanking sequences and HygR/214-6bis for the 3' flanking sequences. One ∆mus51::phleoR ΔPaCdk5::hygR transformant was crossed with the wild type and homokaryotic [phleoS hygR] ΔPaCdk5::hygR progeny was selected for further studies and analyzed by Southern blotting to confirm deletion of PaCdk5 (Figure S1).

Deletion of PaNip30

The PaNip30 (Pa_6_9010) predicted CDS was deleted by a PCR-based strategy [START_REF] Lalucque | A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina[END_REF].

To construct the deletion cassette of the entire PaNip30 coding sequence, a nourseothricin resistance (nouR) marker was fused by PCR with the upstream or the downstream sequence of the PaNip30 gene (Figure S1). 514 bp upstream and 484 bp downstream of PaNip30 were amplified from wild-type genomic DNA with primers 9010_1 and 9010_2 for the upstream region and primers 9010_5 and 9010_6 for the downstream region (Table S1). Primers 9010_2 and 9010_5 contained additional bases allowing fusion with the nouR cassette. The nouR cassette was amplified using primers 9010_MKF/9010_MKR from pBC-nour plasmid [START_REF] Lalucque | A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina[END_REF]. In a second step, two amplification reactions were performed using 9010_1/9010_MKR and 9010_MKF /9010_6 primers pairs to obtain the fusion of two PCR products, the upstream (5') region/nouR and nouR/downstream (3') region. The mixture of these two PCR products was used to transform a ∆mus51::phleoR strain (El- [START_REF] El-Khoury | Gene deletion and allelic replacement in the filamentous fungus Podospora anserina[END_REF]. All the transformants exerted a wild-type phenotype (Figure 1). For twelve of them, integration of the deletion K7 was checked by PCR using primers 9000_10/validMK5' for the 5' flanking sequences and validMK3'/10_20_2R for the 3' flanking sequences. One ∆mus51::phleoR ΔPaNip30::nouR transformant was crossed with the wild type and homokaryotic [phleoS nouR] ΔPaNip30::nouR progeny was selected for further studies and analyz ed by Southern blotting to confirm deletion of PaNip30 (Figure S1).

Double deletion of PaCdk5 and PaNip30

The 5' hygR and 3'hyR split-marker deletion cassettes designed to target PaCdk5 (described above) have been introduced into a purified [phleoR nouR] Δmus51::phleoR ΔPaNip30::nouR homokaryotic strain by transformation. All The [hygR] transformants exerted a [ΔPaCdk5] mutant phenotype (Figure 1). Six transformants were selected and integration of the hygR cassette was checked by PCR using primers 9000_5/HygL for the 5' flanking sequences and HygR/validMK5' for the whole intergenic region in between PaCdk5 and PaNip30 deleted CDS as the HygR primer hybridises in the hygR deletion cassette of ΔPaCdk5::hygR and validMK5' hybridises in the nouR deletion cassette of ΔPaNip30::nouR. Three Δmus51::phleoR ΔPaCdk5::hygR ΔPaNip30::nouR transformants were crossed with the wild type and homokaryotic [phleoS hygR nouR] progeny were selected for further studies.

Proper deletion of both genes was verified by Southern blotting (Figure S1) and the PCR product corresponding to the amplification of the intergenic region (using primers HygR/validMK5') was sequenced, further confirming the integrity of the intergenic space (data not shown).

Deletion of PaTim54

The PaTim54 (Pa_6__9020) predicted CDS was deleted by a split-marker strategy [START_REF] Lalucque | A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina[END_REF]. To construct the deletion cassette of the entire PaTim54 coding sequence, a hygromycin B resistance (hygR) marker was fused by PCR with the upstream or the downstream sequence of the PaTim54 gene (Figure S1). 1102 bp upstream and 832 bp downstream of PaTim54 were amplified from the wild-type genomic DNA with primers 9020_3 and 9020_4 for the upstream region and 9020_1 and 9000_2 for the downstream region (Table S1). Primers 9020_2 and 9020_3 contained additional bases allowing fusion with the hygR cassette. The hygR cassette was amplified using primers 9020_HYGF/9020_HYGR from pBC-hyg plasmid as previously described. In a second step, two amplification reactions were performed using primers 9020_1/HY and YG/9020_4 to obtain the fusion of two PCR products, the upstream (5') region/hygR and hygR/downstream (3') region. The mixture of these two PCR products was used to transform a ∆mus51::su8-1 strain (El-Khoury et al., 2008, p.). All the transformants exerted wild-type phenotype (data not shown). For twelve of them, integration of the deletion K7 was checked by PCR using primers HyGR/9020_5 for the 5' flanking sequences and 9010_2F/HygL for the 3' flanking sequences. Two ∆mus51::su8-1 ΔPaTim54::hygR transformants bearing PCR-verified hygR deletion cassette were crossed with the wild type.

However, no homokaryotic [hygR] progeny was obtained. Further evidences argued that these two primary transformants were heterokaryotic and that deletion of PaTim54 is lethal (see results).

Plasmids

The pBC-hyg and pBC-phleo have been described in [START_REF] Silar | Two new easy to use vectors for transformations[END_REF]. Cosmids of the P. anserina genomic S1) (GoTaq, Promega). The PaTim54 PCR PCR fragment encompassing the 504 base pairs upstream of PaTim54 CDS, the PaTim54 CDS as well as the 182 base pairs downstream of the CDS was generated from wild-type genomic DNA using primers 9020_6F/9020_5R (Table S1). Each PCR fragment was co-transformed with pBC-hyg into PaTim54 2218 with a 3:1.

RNA isolation and RT-qPCR

Vegetative cultures for RNA preparation were performed on Petri dishes containing standard M2 minimal medium and covered with a cellophane sheet. These cultures were inoculated with 6 implants from mat -wild-type strain or mutants. Dishes were placed at 27°C under standard light and were removed from the incubation room at 48 h. Biological materials were collected and flash-frozen in liquid nitrogen. Total RNA of P. anserina was extracted using the RNeasy Plant Mini Kit (cat#74904, Qiagen, Hilden, Germany), including a grinding process using a tissue lyser II (QIAGEN) and a DNase treatment on column. The total RNA quality was checked on gel after standard electrophoresis and quantity was determined by using a Nanodrop spectrophotometer (Nanodrop Technologies). 4 µg of total RNA was used for the RT; reverse transcription was performed with Superscript III or RevertAid (Thermo Fisher Scientific). The 9000RTF and 9000RTR primers for the qPCR on Pa_6_9000/PaCdk5 are located within the CDS at position +723 and +922 respectively. The 9020RTF and 9020RTR primers for the qPCR on Pa_6_9020/PaTim54 are located at position +62 (overlapping the single encoded intron) and position +359 respectively. To quantify the genes with introns, all primers were designed against two consecutive exons (Table S5), and an NRT (Not Reverse Transcribed) control was performed on a pool of replicates to ensure that the Cq was above the Cq obtained from corresponding reverse transcribed RNAs. Each RT-qPCR experiment contained at least three biological replicates (n = 6 for the PaTim54 2218 mutant) (Table S6 andS7), and each biological replicate was analyzed with three technical triplicates. Normalization genes were selected from a pool of 7 housekeeping genes using geNorm (Table S6) [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. RT-qPCR normalization according to the ΔΔCq method [START_REF] Pfaffl | Relative expression software tool (REST©) for groupwise comparison and statistical analysis of relative expression results in real-time PCR[END_REF], standard error and 95% confidence interval calculations, and statistical analyses were performed using REST 2009 software (Qiagen, Hilden, Germany). Table S8 shows fold-changes and standard errors. Genes were defined as downregulated in the mutant strain if the ratio of their transcript level in the mutant strain compared with that in the wild-type strain showed a fold change > 0 and < 1 and with a p-value of < 0.05. On the other hand, genes were defined as up-regulated in the mutant strain if the ratio of their transcript level in the mutant strain compared with that in the wild-type strain showed a > 1 fold change and with a p-value < 0.05. Ratios with a 95 % confidence interval, including 1, were not considered significant [START_REF] Du Prel | Confidence Interval or P-Value?[END_REF].

DNA isolation and qPCR for mtDNA quantification

DNA was prepared as mentioned before [START_REF] Lecellier | Rapid methods for nucleic acids extraction from Petri dish-grown mycelia[END_REF]). An additionnal RNAse step was added requiring a second phenol-chloroform extraction step before final DNA precipitation. For the senDNA assay (Figure 7B), DNA from the senescent apex of a WT strain grown 15 days on M2 medium was extracted. The mitochondrial DNA was quantified with primers targeted to NAD4 gene and normalized to three nuclear loci corresponding to genes PaHMG8, PaHMG9 and MFM.

Importantly, NAD4 was chosen as the target because its chromosomal location is away from the main chromosomal rearrangement leading to senDNA [START_REF] Cummings | The complete DNA sequence of the mitochondrial genome of Podospora anserina[END_REF]. Each qPCR experiment contained four biological replicates and each biological replicate was analyzed with three technical replicates. The mitochondrial copy number normalization was performed according to the ΔΔCq method [START_REF] Pfaffl | Relative expression software tool (REST©) for groupwise comparison and statistical analysis of relative expression results in real-time PCR[END_REF]) by using nuclear loci normalization genes. Standard error and 95% confidence interval calculations, and statistical analyses were performed using REST 2009 software (Qiagen, Hilden, Germany) [START_REF] Pfaffl | Relative expression software tool (REST©) for groupwise comparison and statistical analysis of relative expression results in real-time PCR[END_REF]. Table S10 shows fold-changes and standard errors.

Microscopic analyses

Microscopic observations were made on 3 to 5 days old mycelia grown directly on agar. One cm 2 agar plugs were deposited onto a coverslip and mounted in water supplemented with DAPI (4',6-Diamidine-2'-phenylindole dihydrochloride ; Sigma-Aldrich) 0.1 µg.mL -1 . For pictures of confrontation between P. anserina and P. chrysogenum, plates were co-inoculated, incubated 3 days at 27°C and agar plugs at the confrontation between both species were cut and mounted as previously described but in water. Pictures were taken with an inverted microscope Leica DMI6000; sCMOS camera; LED EL6000 ; dichroic filters XF131 (DAPI) and Qmaxgreen (GFP); Imagoseine Imaging Facility: https://imagoseine.ijm.fr/676/accueil.htm . Pictures were analyzed with Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF].

Diameter of nucleoids and of mitochondria was manually determined on undeconvoluted 2D images using the Fiji software. The size (diameter) of 20 nucleoids (n=20) and the diameter of 20 mitochondria (n=20) has been determined on two to five representative fields of view (pictures) depending on the genotype analyzed. Indeed, since the number of nucleoids and of mitochondria was lower in the +, mito-GFP and the PaTim54 2218 pBC-phleo-PaTim54 mito-GFP strains than in the PaTim54 2218 mito-GFP strain, more samples (pictures) were required to count the 20 nucleoids and the 20 mitochondria in both former strains. GFP fluorescence intensities in the mito-GFP control strain and in the PaTim54 2218 mito-GFP strain were determined using the Fiji software as followed:

The maximum fluorescence intensity for five different transversal cuts along hyphae in teen different samples (pictures) was measured using the "plot profile" function of the Fiji software. For every measure, the background fluorescence intensity was subtracted. The different samples were illuminated in the same conditions with the same exposure time. A t-test on the global distributions (n=50) was performed.

Hyphal Interference assay

Hyphal Interference assays were performed as described in [START_REF] Silar | Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant[END_REF] and adapted from [START_REF] Munkres | Histochemical detection of the secretion of superoxide radicals and hydrogen peroxide by age-1 mutants of Neurospora[END_REF]. Plates were incubated in parallel and in duplicate 3 days at 27°C on standard M2 medium. P. anserina and P. chrysogenum were co-inoculated at an approximate distance of 1.2 cm. For both DAB (diaminobenzidine, Sigma Aldrich) as well as Blue Evans (Sigma Aldrich) staining procedures, the solution was spread with a glass rod to ensure homogenous staining of thalli. For DAB staining (peroxide detection), plates were flooded in 100mM potassium phosphate buffer pH 6.9, 0.9 g.L -1 DAB and incubated 30 min at 27°C. The DAB staining solution was then discarded and pictures were taken 3-6h later. For Blue Evans staining (cell death assay), plates were flooded in 0.1% Evans Blue in water, incubated for 10 min and extensively rinsed with distilled water before pictures were taken.

Longevity assay

For each analyzed genotype, triplicate cultures from three different freshly germinated homokaryotic ascospores of each mating type were incubated on standard M2 medium in longevity tubes (30 cm long) at 27°C. These ascospores were obtained from crossings of the wild type S strain with itself, with PaTim54 2218 and with PaTim54 2218 pBC-phleo-PaTim54. To minimize inheritance of the senescence factor, the wild type S strain was used as female. For this purpose, the "male" implants were removed from the crossings plates before ascospores projections. Total growth distance after 191 days incubation was measured. t-test statistical analysis was performed to compare the growth distance mean of every tested genotypes. Note that measurements of mat-and mat+ strains of each genotype were pooled together for t-test analysis making a pool of 18 measures per genotype.

Mycelium fragmentation

A small plug of mycelium grown on agar plate is grinded is 500 µL sterile water in a FastPrep (500 rpm; 20 sec). The mixture is poured on an agar plate and individual hyphae are harvested under binocular and transferred on standard M2 medium. Homokaryosis is determined by testing the mating type of the regenerated thalli.

Senescent DNA (senDNA) assay

The senDNA assay was performed on the DNA samples used for the qPCR-based mtDNA quantification. DNA from the following genotypes: WT, PaTim54 2218 and PaTim54 2218 pBC-phleo-PaTim54 (two of the four DNA replicates of each genotype were analyzed) as well as DNA sample from an aged WT culture were digested by HaeIII, ran on a 0.8% agarose gel and stained with Ethidium Bromide. SenDNA appear on the gel as intense and discrete bands in aged cultures only [START_REF] Lorin | Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina[END_REF]. Molecular Weight marker: ThermoFisher scientific Generuler TM 1 Kb DNA ladder.

Results

The PDC 2218 mutant

The PDC 2218 strain (all the strains used and generated in this study are reported Table 1) was isolated in a screen designed to identify mutants of Crippled Growth (CG), an epigenetically-regulated degeneration observed when P. anserina is grown on specific media [START_REF] Haedens | Genetic control of an epigenetic cell degeneration syndrome in Podospora anserina[END_REF]. The "Promote Development of Crippled Growth" PDC 2218 was isolated as a "weak" PDC mutant because the severity of the degeneration observed on M2 standard medium was moderate. Additionally, reversion of the degeneration was not clear [START_REF] Haedens | Genetic control of an epigenetic cell degeneration syndrome in Podospora anserina[END_REF]. Compared to the wild type, when grown on M2 standard medium at 27°C, the PDC 2218 mutant grew slower, exhibited a darker green pigmentation and was almost devoid of aerial hyphae (Figure 1 and2). This vegetative phenotype allowed us to characterize the PDC 2218 mutation during positional cloning of the mutation. In addition, PDC 2218 showed temperature sensitivity. The mutant grew much slower than the wild type at 18°C and did not grow at all at 37°C (Figure 2B).

We have previously shown that when P. anserina was challenged with P. chrysogenum, the Hyphal Interference outcome is an oxidative burst on the thallus of P. anserina at the confrontation (revealed by DAB staining) as well as cell death in Penicillium chrysogenum (revealed by Trypan blue or Evans blue staining) [START_REF] Silar | Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant[END_REF]. Remarkably, the PDC 2218 mutant exhibited a modified Hyphal Interference (HI) when confronted with P. chrysogenum with higher DAB precipitation and decreased Evans blue staining (Figure 1) [START_REF] Silar | Hyphal Interference: Self Versus Non-self Fungal Recognition and Hyphal Death[END_REF]. Hence, in the mutant, a huge oxidative burst was triggered during HI but this was not correlated to increased cell death in the competitor (i.e., P. chrysogenum). On the contrary, P. chrysogenum cell death was reduced.

Positional cloning of the PDC 2218 mutation

Previous studies determined that the PDC 2218 mutation had a Second Division Segregation (SDS) frequency of 85%, indicating that it was located in the distal region of the arm of one of the seven chromosomes of P. anserina [START_REF] Haedens | Genetic control of an epigenetic cell degeneration syndrome in Podospora anserina[END_REF]. By crossing the PDC 2218 mutant with different strains bearing mutations in distal parts of chromosomes, we did find a genetic linkage (d=7 cM) between the PDC 2218 mutation and the ΔPaTrx2 deletion [START_REF] Malagnac | PaTrx1 and PaTrx3, Two Cytosolic Thioredoxins of the Filamentous Ascomycete Podospora anserina Involved in Sexual Development and Cell Degeneration[END_REF] (Table S2). In agreement with the 85% SDS of the PaTrx2 gene, this result indicated that the PDC 2218 mutation was located close to PaTrx2 on the left arm distal part of chromosome six. Note that we finally localized the PDC 2218 mutation about 93 kbp from the PaTrx2 gene (Figure 3) (see below). We continued positional cloning of the mutation by crossing the PDC 2218 mutant (generated in the big S strain) with the highly polymorphic strain T of the Podospora comata species [START_REF] Boucher | Species Delimitation in the Podospora anserina/ p. pauciseta/p. comata Species Complex (Sordariales)[END_REF]. We then analyzed co-segregation of polymorphic genetic markers and the PDC 2218 mutation in the progeny. This allowed us to narrow down the location of the PDC 2218 mutation to a 370,822 bp interval delimited by the 214-1 and Yeti-1 molecular markers (Figure 3). Thirteen cosmids of the P. anserina genome bank [START_REF] Espagne | The genome sequence of the model ascomycete fungus Podospora anserina[END_REF] bearing wild-type sequences as well as the hygromycin B resistance gene and overlapping the candidate interval were transformed into the PDC 2218 mutant (see materials and methods). Only transformation with the 11G02 cosmid gave 12 "wild-typelooking" [WT] (from now on, phenotypes are indicated in square brackets) out of 34 [hygR] transformants, suggesting that 11G02 carried the wild-type allele of the gene mutated in PDC 2218 . It also indicated that the PDC 2218 mutation was recessive. The 22 non-complementing transformants likely resulted from the heterologous integration of the cosmid and fortuitous inactivation of the complementing sequence. Transformation with the partially overlapping 31C04 cosmid did not provide any complemented transformant. This led us to conclude that the PDC 2218 mutation was in the non-overlapping part of the sequence carried by the 11G02 cosmid, allowing us to narrow down the candidate interval for the mutation (Figure 3).

Seven predicted CDS were encoded in this interval. We selected seven plasmids of the P. anserina genome bank corresponding to the seven genes, each plasmid bearing a single predicted CDS (see materials and methods). Since the pCDNA.2 backbone of the bank's plasmids has no fungal selective marker, co-transformation of each plasmid along with empty pBC-hyg plasmid conferring resistance to hygromycin B was performed. Complementation of the mutant phenotype was observed both when wild-type Pa_6_9000 (pPaCdk5 plasmid) or Pa_6_9020 CDS (pPaTim54 plasmid) were introduced into PDC 2218 (Figure 3). The Pa_6_9000 predicted CDS encodes Pho85/PaCdk5, the P. anserina homologue of the yeast Cyclin Dependent Kinase 5 [START_REF] Huang | Pho85, a multifunctional cyclin-dependent protein kinase in budding yeast[END_REF] and Pa_6_9020 encodes the homologue of the yeast mitochondrial Translocase of the Inner Membrane protein Tim54p, PaTim54 (Figure S2) [START_REF] Kerscher | The Tim54p-Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane[END_REF] transgenes were co-integrated in the genome as often observed in P. anserina [START_REF] Razanamparany | Non-homologous integration of transforming vectors in the fungus Podospora anserina: sequences of junctions at the integration sites[END_REF] and that the ΔPaCdk5 ΔPaNip30 pPaCdk5 pBC-phleo progeny presented wild-type phenotype. These data argued that only deletion of PaCdk5 accounted for the mutant vegetative phenotype in ΔPaCdk5 as well as in ΔPaCdk5 ΔPaNip30. HI was as in the wild type for the three K.O strains tested: ΔPaCdk5, ΔPaNip30 and ΔPaCdk5 ΔPaNip30 (Figure 1). In conclusion, PaNip30 has no role in the processes studied here and PaCdk5 loss-of-function did not account for the PaTim54 2218 phenotypes.

PaCdk5 does not trans-complement PaTim54 2218

The absence of mutation in PaCdk5 CDS, its unaffected expression in PaTim54 2218 as well as the phenotype of ΔPaCdk5 were in sharp contrast to the observed complementation of PaTim54 2218 when this latter was transformed with PaCdk5 wild-type copy bearing transgenes. This led us to hypothesize that the integration sites of the transgenes might account for this discrepancy. We thus 1). This result was intriguing for bona fide functional complementation relies on "in trans" rescue of the loss-of-function of a gene by its wild-type copy. We hence addressed the question whether pPaCdk5 could trans-complement PaTim54 2218 . In a first step, three independent [WT] complemented ΔPaCdk5 pPaCdk5 pBC-phleo strains were crossed with the wild type (Table 3). In the progeny of the three crossings, [hygR phleoR] spores presented a [WT] phenotype while the [hygR phleoS] progeny conserved a mutant [ΔPaCdk5] phenotype. In addition, equal distribution of the four different phenotypic categories showed genetic independence between both loci, leading us to conclude to ectopic chromosomal co-integration of both pPaCdk5 and pBC-phleo (χ 2 analysis: P>0.05).

We then tested if these transgenes could trans-complement PaTim54 2218 by crossing one ΔPaCdk5 pPaCdk5 pBC-phleo F1 progeny from each parent transgenic line with PaTim54 2218 (Table 4). S3). Progeny analysis for both transformants showed that, first, pPaCdk5 and pBCphleo were ectopically co-integrated (no genetic linkage between the PaCdk5/PaTim54 2218 locus and the transgenes integration sites; χ 2 analysis: P>0.05) and second that the pPaCdk5 transgenes previously shown as unable to complement PaTim54 2218 did complement ΔPaCdk5. Indeed, in both crossings, the ΔPaCdk5 pPaCdk5 pBC-phleo progeny presented wild-type phenotype. In addition, one [PaTim54 2218 ] PaTim54 2218 pPaCdk5 pBC-phleo transformant was crossed to the double ΔPaCdk5 ΔPaNip30 mutant (Table S4). In the progeny, the ΔPaCdk5 ΔPaNip30 pPaCdk5 pBC-phleo descendants showed wild-type phenotype, demonstrating as mentioned before that only PaCdk5 loss-of-function was responsible for the [pPaCdk5] mutant phenotype in ΔPaCdk5 ΔPaNip30.

Altogether, these data argued that the PaTim54 2218 mutation was not functionally transcomplemented by wild-type pPaCdk5 but rather genetically cis-suppressed by genetically linked pPaCdk5-bearing transgenes (Figure 1). Importantly, the PaCdk5 PCR fragment allowing cis-suppression of PaTim54 2218 mutation in the co-transformation experiments mentioned above does not span the PaTim54 2218 deletion (Figure 3).

PaTim54 is the gene affected in

PaTim54 is essential in P. anserina

[hygR] mat+/mat-ΔPaTim54/+ self-fertile dikaryotic ascospores, (see above) to determine the mating type of the nucleus carrying the ΔPaTim54 deletion. To this purpose, we crossed one mat+/mat-ΔPaTim54/+ with both the mat+ and the mat-wild-type strains. As shown in Table 6, only crossing with the mat-wild type partner yielded SDS asci with four ΔPaTim54/+ spores, arguing that in the analyzed mat+/mat-ΔPaTim54/+ strains, the ΔPaTim54 deletion was carried by the mat+ nucleus (Table 6). Then, we crossed the [hygR] mat+/mat-ΔPaTim54/+ strain carrying the ΔPaTim54 deletion in the mat+ nucleus with a [WT phleoR] mat-PaTim54 2218 pBC-phleo-PaTim54 strain (Table 7). In the progeny, we harvested [WT hygR phleoR] viable homokaryotic ΔPaTim54 pBC-phleo-PaTim54 as well as viable dikaryotic ΔPaTim54/ΔPaTim54 pBC-phleo-PaTim54/+ descendants.

Notably, the χ 2 statistical analysis did not indicate genetic linkage between PaTim54 and the different pBC-phleo-PaTim54 transgenes studied (P>0.05). When tested for HI, the ΔPaTim54 pBC-phleo-PaTim54 progeny was comparable to the wild type (Figure 1). These data confirmed that wild-type PaTim54 fully trans-complemented ΔPaTim54. Finally, to test whether PaTim54 was essential in P. ΔPaTim54. This latter result strongly argued that deletion of PaTim54 was lethal and more specifically that PaTim54 was essential for mycelial growth in P. anserina.

PaTim54 gene expression is affected in PaTim54 2218

Because the sequence of the PaTim54 CDS was not altered in PaTim54 2218 , we hypothesized that its expression may be affected in the mutant. To address this question, we tested PaTim54 gene expression by RT-qPCR. Strikingly, PaTim54 mRNA level was less than 10% of the wild-type level in Altogether, these data strongly argued that only very low PaTim54 gene expression level was responsible for the pleiotropic [PaTim54 2218 ] mutant phenotypes.

PaMpk1 regulates PaTim54 2218 -triggered oxidative burst

As shown in Figure 1, we could detect a massive oxidative burst at the confrontation between PaTim54 2218 and P. chrysogenum during HI. However, cell death at the confrontation in P. chrysogenum revealed by Evans blue staining was abolished compared to that when the wild type was challenged with P. chrysogenum. The PaMpk1 MAP Kinase pathway as well as the PaNox1 complex are important regulators of HI in P. anserina. Indeed, the oxidative burst along with cell death in P. chrysogenum at the confrontation are both abolished when ΔPaMpk1 and PaNox1 - mutants are challenged (Figure 1) [START_REF] Silar | Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant[END_REF]. In order to determine whether PaMpk1 and PaNox1

PaTim54 2218 mutation affects the mitochondrial network

With the aim to investigate the mitochondrial network in PaTim54 2218 when grown on standard medium but also when challenged with P. chrysogenum, we constructed a PaTim54 2218 mito-GFP [START_REF] Sellem | A mutation in the gene encoding cytochrome c1 leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina[END_REF]. In yeast, Tim54p is involved in the import of proteins of the inner membrane and it has been shown that null mutation of tim54 does not affect import of soluble matrix proteins [START_REF] Kerscher | The Tim54p-Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane[END_REF]. Accordingly, GFP fluorescence intensities measured in the PaTim54 2218 mito-GFP strain were not statistically different than in the mito-GFP control strain arguing for a normal import of the soluble matrix mito-GFP reporter protein in PaTim54 2218 mutant background (Figure S3). To standardize our cytological analyses, we focused on hyphae of the growing edge of the thallus. Indeed, mitochondrial morphology is highly dynamic, with mitochondria forming long longitudinal tubes in the apical hyphae (thread-like network) to shorter tubes and finally rounded mitochondria in the central part of the thallus (data not shown). We thus analyzed GFP tagging pattern at the edge of the thallus in the PaTim54 2218 mito-GFP tagged strain and we compared it to the control strain bearing only the mito-GFP transgene as well as to the complemented PaTim54 2218 pBC-phleo-PaTim54 mito-GFP strain. Green fluorescence pattern in the control strain as well as in the complemented PaTim54 2218 pBC-phleo-PaTim54 mito-GFP strain exhibited a thread-like mitochondrial network formed of highly tubular and easily trackable mitochondria (Figure 5A). On the contrary, GFP tagging pattern in PaTim54 2218 mito-GFP was much more intricate. Mitochondria in the mutant looked shorter, thinner, and somehow packed, as if filling the cytoplasm. They did not form long tubular structures as in the control and in the complemented strain. In addition to the size difference, we also determined that their diameter was significantly smaller (334 ± 37 nm; t-test: P<10 -9 ) in PaTim54 2218 compared to the control (456 ± 52 nm) and the complemented strain (418 ± 60 nm; t-test: P<10 -5 ).

We also analyzed the mitochondrial network at the edge of the thallus when P. anserina was challenged with P. chrysogenum. For both the mito-GFP control strain as well as for PaTim54 2218 mito-GFP, tagging pattern in hyphae in close contact to P. chrysogenum was not different from the tagging pattern in hyphae of the growing edge without challenge (Figure 6). This indicated that challenging with P. chrysogenum did not significantly affect P. anserina mitochondrial network at the confrontation. However, we often observed small lateral branching hyphae that swelled in PaTim54 2218 (Figure 6). When laterally branching hyphae were observed in the control, they exhibited standard morphology but no swelling. This latter observation indicated that the challenge with P. chrysogenum did induce swelling of small growing hyphae specifically in PaTim54 2218 .

PaTim54 2218 mutation affects nucleoids

In S. cerevisiae, it has been shown that the number of nucleoids, the structure where the mitochondrial DNA (mtDNA) is found, was decreased in the tim54 mutant strains [START_REF] Hwang | Tim54p connects inner membrane assembly and proteolytic pathways in the mitochondrion[END_REF]. In order to assay mtDNA stability in P. anserina, we quantified mtDNA by qPCR and we cytologically analyzed the distribution of nucleoids on young mycelium. Samples were stained with DAPI and the number of nucleoids in 10 µm length hyphal segments was quantified (Figure 5A). Surprisingly, the number of nucleoids in PaTim54 2218 mito-GFP was dramatically increased compared to the mito-GFP control (32 ± 9 in PaTim54 2218 vs. 9 ±3 in the control; t-test: P<10 -9 ) and to the PaTim54 2218 pBC-phleo-PaTim54 mito-GFP complemented strain (12 ± 4; t-test<10 -8 ) (Figure 5B).

Interestingly, these nucleoids were significantly smaller in PaTim54 2218 mito-GFP than in the control (313 ± 72 nm in PaTim54 2218 vs. 429 ± 75 nm in the control; t-test: P<10 -4 ) and in the complemented strain (416 ± 53 nm; t-test<10 -4 ) (Figure 5C). These observations suggested that the nucleoids in PaTim54 2218 mito-GFP were somehow disorganized and likely fragmented. However, these data did not allow us to conclude on the stability of the mtDNA in PaTim54 2218 . To this aim, the ratio of mtDNA versus nuclear DNA in young mycelium was measured by qPCR in PaTim54 2218 , PaTim54 2218 pBC-phleo-PaTim54 and the wild type (Figure 7A). The level of mtDNA was similar in the three tested strains, leading us to conclude that the important accumulation of nucleoids of small size in PaTim54 2218 was indicative of disorganization and/or fragmentation of nucleoids but not of increased mtDNA level. Importantly, we determined that senescent DNA (senDNA) was absent in the DNA samples used for the mtDNA level quantification (Figure 7B), which is in agreement with the paradigm that senDNA is absent in young mycelium [START_REF] Osiewacz | Mitochondrial pathways governing stress resistance, life, and death in the fungal aging model Podospora anserina[END_REF]. This suggested that senDNA did not account for disorganized and/or fragmented nucleoids observed in PaTim54 2218 young mycelium and this indicated that senDNA did not alter mtDNA level quantifications. Finally, the fact that the number of nucleoids, their size as well as the quantity of mtDNA in the PaTim54 2218 pBC-phleo-PaTim54 complemented strain were similar to the wild type, emphasized that all the defects evidenced above in PaTim54 2218 are due to the PaTim54 2218 hypomorphic allele.

PaTim54 2218 mutation affects the PaAOX gene expression level and longevity in P. anserina

P. anserina is a potent model system to study aging [START_REF] Marcou | Notion de longévité et nature cytoplasmique du déterminant de la senescence chez quelques champignons[END_REF][START_REF] Osiewacz | Mitochondrial pathways governing stress resistance, life, and death in the fungal aging model Podospora anserina[END_REF]. Indeed, contrary to many filamentous fungi, P. anserina vegetative growth is limited to about 10 cm (about 15 to 20 days of growth) on standard M2 medium and at standard temperature (27°C). This phenomenon is known as senescence (Rizet, 1953b). To study senescence in the wild type, in PaTim54 2218 as well as in the PaTim54 2218 pBC-phleo-PaTim54 complemented strain, we analyzed the growth distance of mycelium cultures from freshly germinated spores (Figure 8). In these longevity assays, all the cultures of the wild type as well as of the PaTim54 2218 pBC-phleo-PaTim54 complemented strains died after an average of 10 cm of growth, corresponding to about 15-20 days of longevity. On the contrary, 12/18 cultures of the PaTim54 2218 genotype and corresponding to three replicates from three single mat+ ascospores and one single mat-ascospore were still growing after 191 days of incubation for a total growth distance of approximately 1 m, a longevity never observed in WT. Statistical analysis supported the difference observed between the PaTim54 2218 strain and the WT or the PaTim54 2218 pBC-phleo-PaTim54 complemented strains (Figure 8; P<4.10 -8 ). Worth is to note that these cultures were still growing when measurements were stopped. Remarkably, the three replicates from two individual mat + PaTim54 2218 spores underwent senescence after an average of 22 and 25 cm growth distance respectively. This indicated that senescence was not completely abolished in PaTim54 2218 . Nevertheless, these longevity assays demonstrated that PaTim54 2218 behaved as a long-lived mutant and that PaTim54 2218 hypomorphic allele was responsible for increased longevity. P. anserina is an obligate aerobe fungus in which respiratory mutants lacking Cytochrome c oxidase activity (E.g PaCOX1 and PaCOX5 mutants) are nonetheless viable but they astonishingly show strikingly increased lifespan. In such mutants, overexpression of the alternative oxidase encoding gene PaAOX has been shown to compensate for the lack of Cytochrome c oxidase activity and the exclusive use of the alternative respiration pathway that produces less reactive oxygen species leads to increased lifespan [START_REF] Dufour | A causal link between respiration and senescence in Podospora anserina[END_REF][START_REF] Lorin | Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina[END_REF][START_REF] Sellem | A mutation in the gene encoding cytochrome c1 leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina[END_REF]. In order to test

Discussion

The PDC 2218 mutant and Hyphal Interference

Microbial communities are complex ecosystems where interplay between fungi, bacteria and invertebrates such as nematodes are of major importance [START_REF] Crowther | Functional and ecological consequences of saprotrophic fungus-grazer interactions[END_REF][START_REF] De Boer | Living in a fungal world: impact of fungi on soil bacterial niche development⋆[END_REF]. Interactions can be of different kinds and in this paper we focused on Hyphal Interference (HI), a defense response shown by filamentous fungi when their hyphae directly contact a competitor fungus. This implies that fungi capable of HI i) can sense the competitor's hyphae, ii) recognize them as non-self and iii) are endowed with an appropriate downstream signaling pathway and effectors required to trigger the HI response. The ascomycete fungus P. anserina is able to display a HI response when challenged with various fungal competitors; however the basidiomycete Coprinopsis cinera is able to kill P. anserina hyphae, while P. anserina is able to kill P. chrysogenum [START_REF] Silar | Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant[END_REF].

Non-self-recognition not only in P. anserina but more generally in the fungal kingdom is largely unknown. First involved in cell death during Vegetative Incompatibility in P. anserina and in N. crassa, Nucleotide Oligomerization Domain (NOD)-like Receptors (NLRs) could play the role of Pattern Recognition Receptors (PRRs) in fungi [START_REF] Bidard | The Transcriptional Response to Nonself in the Fungus Podospora anserina[END_REF][START_REF] Lamacchia | Overlapping Podospora anserina Transcriptional Responses to Bacterial and Fungal Non Self Indicate a Multilayered Innate Immune Response[END_REF][START_REF] Paoletti | Vegetative incompatibility in fungi: From recognition to cell death, whatever does the trick[END_REF][START_REF] Uehling | Do fungi have an innate immune response? An NLR-based comparison to plant and animal immune systems[END_REF]. Nowadays, only Het-C in N. crassa, "the host", has been shown as a true PRR able to bind the PhcA protein from Pseudomonas syringae, the "pathogen", and to trigger cell death in the host [START_REF] Wichmann | A novel gene, phcA from Pseudomonas syringae induces programmed cell death in the filamentous fungus Neurospora crassa[END_REF]. How P.anserina recognizes non-self-hyphae is an open question. Similarly, it is not clear how cell death in the challenger, here P. chrysogenum, is triggered.

Fungi are able to produce many kinds of toxic compounds in order to kill their competitors. Among the list of active antifungal compounds able to trigger hyphal death are secondary metabolites, volatile organic compounds, small secreted polypeptides and secreted lytic enzymes [START_REF] Silar | Hyphal Interference: Self Versus Non-self Fungal Recognition and Hyphal Death[END_REF]. A burst of Reactive Oxygen Species (ROS) is generated on the thallus of P. anserina when challenged by different fungal species. However, these ROS are unlikely directly responsible for P. chrysogenum cell death. Indeed, the PDC 2218 mutant analyzed here shows an increased oxidative burst and triggers less death in the competitor. On the same line, we have previously shown that the ROS produced by the PaNox1 NADPH oxidase are involved in signaling rather than in killing during HI [START_REF] Brun | Functions and regulation of the Nox family in the filamentous fungus Podospora anserina : a new role in cellulose degradation[END_REF].

Interestingly, epistasis analysis shows that PaNox1 only plays a minor role in the oxidative burst displayed by PDC 2218 . The additional sources of these ROS remain to be identified. Epistasis data indicate that their production in PDC 2218 is under the control of the CWI PaMpk1 MAP kinase pathway.

We want to stress the point that Nox1 and the Mpk1 MAP Kinase pathway control virulence in mycoparasitic Trichoderma sp. developed for biocontrol of fungal plant pathogens as well [START_REF] Druzhinina | Trichoderma: the genomics of opportunistic success[END_REF][START_REF] Karlsson | Necrotrophic Mycoparasites and Their Genomes[END_REF]. For instance, the Nox1 NADPH oxidase of Trichoderma harzanium and the ROS it produces are important in the biocontrol of the phytopathogen Phytium ultimum [START_REF] Montero-Barrientos | Functional Analysis of the Trichoderma harzianum nox1 Gene, Encoding an NADPH Oxidase, Relates Production of Reactive Oxygen Species to Specific Biocontrol Activity against Pythium ultimum[END_REF]. In the closely related species Trichoderma virens, the TmkA (=Tvk1) and TmkB MAP kinases, homologous to PaMpk2 and PaMpk1 respectively, play key roles in the mycoparasitic activity against Sclerotium rolfsii, Rhizoctonia solani and Phytium spp. [START_REF] Kumar | Overlapping and distinct functions of two Trichoderma virens MAP kinases in cell-wall integrity, antagonistic properties and repression of conidiation[END_REF][START_REF] Mukherjee | TmkA, a Mitogen-Activated Protein Kinase of Trichoderma virens, Is Involved in Biocontrol Properties and Repression of Conidiation in the Dark[END_REF]. HI and mycoparasitism may be closely related processes relying on a common set of regulatory pathways.

The

PaTim54, mitochondria and defense response

The PaTim54 gene encodes the orthologue of the yeast mitochondrial Translocase of the inner Membrane Tim54p [START_REF] Kerscher | The Tim54p-Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane[END_REF]. PaTim54 protein shares 60% and 40% identity with N. crassa and S. cerevisiae Tim54p proteins, respectively. Noteworthy, no other homologue has been found in the P. anserina genome. Similarly to the yeast tim54 mutants, PaTim54 2218 shows slow growth at cold temperature (18°C) and thermal sensitivity at 37°C. However, while the yeast Tim54p has no major role in mitochondrial morphology, PaTim54 2218 mutation promotes significant mitochondrial fission. Indeed, cytological analysis of mitochondria in living cells shows that the thread-like mitochondrial network is altered in PaTim54 2218 . This mitochondrial morphology change in PaTim54 2218 is reminiscent of increased mitochondrial fission and/or decreased mitochondrial fusion. In addition in the tim54 yeast mutants, the number of nucleoids is decreased [START_REF] Hwang | Tim54p connects inner membrane assembly and proteolytic pathways in the mitochondrion[END_REF]. Although we have observed that the number of nucleoids is dramatically increased in PaTim54 2218 , mtDNA level remains unchanged. Hence, the PaTim54 2218 allele seems to promote the disorganization and fragmentation of nucleoids but not mtDNA degradation in P. anserina.

A growing body of evidence links mitochondria to immunity. In humans, several immune syndromes are now considered as "likely" due to primary mitochondrial disorders like the Barth Syndrome or the Cartilage Hair Hypoplasia. It becomes clear that mitochondria participate in RLR signaling (an antiviral response based on RLR-Retinoic acid Like Receptor), antibacterial immunity and inflammation [START_REF] Walker | Powering the Immune System: Mitochondria in Immune Function and Deficiency[END_REF][START_REF] West | Mitochondria in innate immune responses[END_REF]. Noteworthy, both the mitochondrial ROS production as well as the capacity of mitochondria to fuse are two important phenomena involved in RLR signaling and inflammation, respectively. Signaling responses to non-self through mitochondria appears thus conserved during evolution. We show here that the P. anserina mitochondrial network is not modified in hyphae in close contact with hyphae of competing fungus during HI. However, we show that PaTim54 function is required for the down-regulation of the oxidative burst during HI and to trigger hyphal cell death in the challenging fungus. Hence, like the PaMpk1 and the PaNox1 signaling pathways, mitochondria appear as important actors of HI. Further studies will be required to understand how mitochondria and the PaMpk1 pathway interact in the regulation of the HI response in P. anserina.

PaTim54, mitochondria and aging

Mitochondria play a major role in aging in a wide range of eukaryotes as well. This organelle is known to provide the cell with ATP through the respiratory chain. However, the byproducts of this process, namely the ROS, are able to damage cellular components such as proteins, nucleic acids and lipids. This damaging capacity of mitochondria-produced ROS is well known as the "Mitochondrial Free Radical Theory of Aging" [START_REF] Harman | The Biologic Clock: The Mitochondria?[END_REF]. However, ROS production is not anymore considered as the only way mitochondria impact cell longevity. Evidences show that several regulatory pathways merging in a quality control system in mitochondria influence lifespan as well [START_REF] Osiewacz | Mitochondrial quality control in aging and lifespan control of the fungal aging model Podospora anserina[END_REF][START_REF] Osiewacz | Mitochondrial pathways governing stress resistance, life, and death in the fungal aging model Podospora anserina[END_REF][START_REF] Osiewacz | Mitochondrial Quality Control: Impact on Aging and Life Span -A Mini-Review[END_REF]. Briefly, this quality control includes ROS scavenging, mtDNA maintenance, protein-repair, -refolding and -degradation, mitochondria shaping (fusion vs. fission) and mitophagy [START_REF] Fischer | Mitochondrial quality control: an integrated network of pathways[END_REF]. In yeast, Tim54p is an integral protein of the mitochondrial inner membrane with two predicted transmembrane domains. Tim54p participates in at least two distinct complexes. On the one hand, it associates with the voltage dependent channel protein Tim22p responsible for the import of integral membrane proteins. Although the role of Tim54p in the complex remains unclear, Tim54p per se has been shown to be required for the insertion/import of the Tim22p translocase, the ATP/ADP carrier Aac1p and the i-AAA protease Yme1p, three intrinsic proteins of the mitochondrial inner membrane [START_REF] Bolender | Multiple pathways for sorting mitochondrial precursor proteins[END_REF][START_REF] Kerscher | The Tim54p-Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane[END_REF][START_REF] Kovermann | Tim22, the Essential Core of the Mitochondrial Protein Insertion Complex, Forms a Voltage-Activated and Signal-Gated Channel[END_REF][START_REF] Kurz | Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway[END_REF]. On the other hand, yeast Tim54p directly interacts with the i-AAA-protease Yme1p [START_REF] Hwang | Tim54p connects inner membrane assembly and proteolytic pathways in the mitochondrion[END_REF]. Tim54p is absolutely required to stabilize the 1M Da high molecular weight Yme1p complexes as well as for the protease activity of the complex. Tim54p therefore plays a pivotal role in the import of proteins into mitochondria as well as in their degradation [START_REF] Hwang | Tim54p connects inner membrane assembly and proteolytic pathways in the mitochondrion[END_REF]. Both roles may account for PaTim54 2218 phenotypes.

We have shown that the PaTim54 2218 mutation increases lifespan, a feature present in many mitochondrial mutants. We have also demonstrated that mitochondrial morphology as well as nucleoids organization are affected in the mutant. Noteworthy in PaTim54 2218 , the increased lifespan may not be correlated to a decrease in mitochondrial fission. Finally, as supported by PaAOX gene overexpression in PaTim54 2218 which is characteristic of lack of Cytochrome c oxidase function [START_REF] Dufour | A causal link between respiration and senescence in Podospora anserina[END_REF][START_REF] Lorin | Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina[END_REF][START_REF] Sellem | A mutation in the gene encoding cytochrome c1 leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina[END_REF], insufficient PaTim54 gene expression may affect the respiratory chain function by disturbing import of its integral components [START_REF] Kerscher | The Tim54p-Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane[END_REF][START_REF] Kovermann | Tim22, the Essential Core of the Mitochondrial Protein Insertion Complex, Forms a Voltage-Activated and Signal-Gated Channel[END_REF]. It has been shown that Yme1p plays a major role in shaping mitochondrial morphology and in mtDNA stability [START_REF] Campbell | Mitochondrial morphological and functional defects in yeast caused by yme1 are suppressed by mutation of a 26S protease subunit homologue[END_REF]. N. crassa IAP-1 and P. anserina PaIAP, both orthologues of Yme1p, have previously been studied [START_REF] Klanner | MAP-1 and IAP-1, two novel AAA proteases with catalytic sites on opposite membrane surfaces in mitochondrial inner membrane of Neurospora crassa[END_REF][START_REF] Weil | Unmasking a temperaturedependent effect of the P. anserina i-AAA protease on aging and development[END_REF]. As in yeast, IAP-1 and PaIAP are involved in proteolysis of misfolded proteins, participating in protein quality control [START_REF] Osiewacz | Mitochondrial quality control in aging and lifespan control of the fungal aging model Podospora anserina[END_REF][START_REF] Osiewacz | Mitochondrial pathways governing stress resistance, life, and death in the fungal aging model Podospora anserina[END_REF]. The N. crassa iap-1 mutant strains show slow growth on non-fermentable carbon sources at high-temperature (40°C) and mutations in PaIAP leads to increased lifespan at normal temperature in P. anserina. Because of the similar lifespan increase in PaTim54 2218 and in PaIAP mutants, it is tempting to speculate that both gene products may also work together in P. anserina. However, both mutant strains exhibit some differences in their phenotypes. For instance, PaTim54 but not PaIAP is essential in P. anserina.

Furthermore, the hypomorphic PaTim54 2218 allele is thermosensitive in contrast to knocking-out PaIAP that slightly increases longevity at 37°C [START_REF] Weil | Unmasking a temperaturedependent effect of the P. anserina i-AAA protease on aging and development[END_REF]. This may be accounted for by a major role of Tim54p in protein import. Further studies of the role of PaIAP in Hyphal Interference as well as thorough studies of the double PaTim54 2218 ΔPaIAP mutant in comparison with both single PaTim54 2218 and ΔPaIAP mutants should give new insights into the role of both integral proteins of the mitochondrial inner membrane.

Conclusion

The characterization of the gene actually affected in the PDC 2218 S6 and S7 for Cq and Table S8 for numerical values of FC). Normalization genes selected with geNorm [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF] were AS1, GPD and UBC, with an average stability value M= 0.346 and V3/4 = 0.093 (see Table S6). Bars show standard errors and stars (*) indicate FC with a p-value < 0.05 (see Table S8 for numerical values of standard errors). FCs with a 95 % confidence interval including the ratio 1 or very close to 1 were not considered as significant, even if the p-value is below the cut-off [START_REF] Du Prel | Confidence Interval or P-Value?[END_REF]. S9 for Cq and Table S10 for numerical values of FC). Three nuclear loci were used for normalization (Table S9). Bars

show standard errors and stars (*) indicate FC with a p-value < 0.05 (see Table S5

  library ref: 27C11, 31C04, 8H08, 11G02, 46G03, 1B05, 44C04, 3H09, 8C03, 38F08, 36C09, 39C12 and 12F01 bearing an hygR selection marker have been used for positional cloning. Plasmids of the P. anserina genomic library ref: 342AG09 (bearing the Pa_6_8980 CDS), 340DH08 (Pa_6_8990 CDS), 224BE12 (Pa_6_8991 CDS), 328CD12 (Pa_6_9000 CDS) renamed pPaCdk5, 193BG01 (Pa_6_9010 CDS) renamed pPaNip30, 434CF12 (Pa_6_9020 CDS) renamed pPaTim54 and 142DC07 (Pa_6_9030 CDS) have been used for positional cloning. The pCDNA.2 plasmid backbone does not bear selection marker in P. anserina. The average size of the insert is 3 kbp (Espagne et al., 2008). 2.7. Constructions 2.7.1. pBC-hyg-PaCdk5: The pPaCdk5 plasmid was digested by the NotI and SpeI restriction enzymes. It generated a 3632 bp fragment bearing the 1023 bp of PaCdk5 predicted CDS plus the 1536 bp upstream and the 1073 bp downstream of PaCdk5 predicted CDS. This insert was subcloned into Not1/SpeI-digested pBC-hyg generating the pBC-hyg-PaCdk5 plasmid. 2.7.2. pBC-hyg-PaTim54 and pBC-phleo-PaTim54: The pPaTim54 plasmid was digested by the SpeI and XhoI restriction enzymes. It generated a 3135 bp fragment bearing a 1450 bp encompassing pPaTim54 predicted CDS (1386 bp) and one intron (64 bp) plus the 1019 bp upstream and the 666 bp downstream of PaTim54 predicted CDS. This insert was subcloned into SpeI/XhoI-digested pBC-hyg or pBC-phleo, generating the pBC-hyg-PaTim54 and the pBC-phleo-PaTim54 plasmids respectively. 2.8. PCR based complementation of PaTim54 2218 : The PaCdk5 PCR PCR fragment encompassing the 152 base pairs upstream of the PaCdk5 CDS as well as the 139 base pairs downstream of the CDS was generated from wild-type genomic DNA using primers 9000_1F/9000_4R (Table

  . A total of 15 [WT] out of 137[hygR] transformants were obtained with the plasmid carrying PaCdk5 and 44 [WT] out of 49[hygR] transformants were obtained with the plasmid carrying PaTim54. On the contrary, co-transformation with the five other selected plasmids or empty pBC-hyg plasmid alone did not promote recovery of [WT] transformants. Noteworthy, co-transformation with the plasmid bearing the predicted PaNip30 CDS (Pa_6_9010), encoding the Nip30 homologue protein of unknown function (Mammalian Gene Collection (MGC) Program Team, 2002), located between PaCdk5 and PaTim54 on the chromosome did not result in any complementation (0 [WT]/ 35 [hygR]). These results were confirmed by cotransformation experiments performed using PCR fragments consisting of PaCdk5 wild-type CDS (PaCdk5 PCR ) or of PaTim54's one (PaTim54 PCR ) with pBC-hyg: 4 [WT] out of 50 [hygR] transformants for PaCdk5 PCR and 88 [WT] out of 97 [hygR] transformants for PaTim54 PCR were observed (see materials and methods and Figure 3). With the aim to rule out any artefactual effect due to cotransformation, we generated the pBC-hyg-PaCdk5 and pBC-hyg-PaTim54 plasmids by cloning the PaCdk5 and the PaTim54 predicted CDS from pPaCdk5 and pPaTim54 into pBC-hyg respectively (see materials and methods and Figure 3). In agreement with the previous experiments, complementation of PDC 2218 was observed with both pBC-hyg-PaCdk5 (15 [WT]/ 50 [hygR]) and pBC-hyg-PaTim54 (47 [WT]/ 50 [hygR]). For the sake of simplicity, the PDC 2218 mutation was renamed PaTim54 2218 in the following parts of the manuscript as PaTim54 proved to be the gene affected in PDC 2218 (see below). Five PaTim54 2218 pBC-hyg-Cdk5 as well as five PaTim54 2218 pBC-hyg-PaTim54 independent transformants presenting wild-type vegetative growth were tested for HI. Every transformants showed wild-type HI indicating that the HI default was complemented as well. Representative result for one of the [WT] transformants tested is shown Figure 1. This transformant of the PaTim54 2218 pBC-hyg-Cdk5 genotype was annotated "cis" (see below for further explanations). Unexpectedly, taken together, these above complementation experiments did not allow us to determine which of PaCdk5 or PaTim54 was affected in the PaTim54 2218 mutant. 3.3. Molecular characterization of the PaTim54 2218 mutation Both PaCdk5 and PaTim54 genes were amplified by PCR in PaTim54 2218 and sequenced. In addition to the CDS per se, sequencing included 458 bp upstream and 139 bp downstream of PaCdk5 CDS as well as 372 bp upstream and 223 bp downstream of PaTim54 CDS. Surprisingly, sequencing of both genes did not reveal any mutation. With the aim to further investigate the sequence of the region between PaCdk5 and PaTim54, we attempted PCR-based chromosome walking within the 3157 bp separating both CDS (Figure 3). Strikingly, PCR amplification of the region between PaCdk5 and PaNip30 (using primers 9000_3F/9010_3R binding in each CDS respectively) was about 1600 bp shorter than expected in the PaTim54 2218 mutant, strongly suggesting a deletion in the mutant's genome. Sequencing of this PCR product confirmed that 1560 bp were lacking in PaTim54 2218 . This deletion removed the first 140 bp of PaNip30 CDS as well as 1420 out of the 1856 bp of the intergenic region between PaCdk5 and PaNip30. Analysis of P. anserina ESTs[START_REF] Espagne | The genome sequence of the model ascomycete fungus Podospora anserina[END_REF] showed that the PaCdk5 gene is transcribed and that it bears a long 3' UTR overlapping the observed deletion in the mutant (Figure3). However, this deletion did not affect the transcription level of PaCdk5, which was similar to the one of PaCdk5 in the wild type (Figure4). This result, together with the absence of mutations in the PaCdk5 predicted CDS in the PaTim54 2218 mutant pointed to a role of PaTim54 in the specific phenotype of the PaTim54 2218 mutant. 3.4. PaCdk5 and PaNip30 loss-of-functions are not responsible for the PaTim54 2218 phenotypes With the aim to test the role of PaCdk5 and PaNip30 in P. anserina, we Knocked Out (K.O) PaCdk5 and PaNip30 separately and we also generated the double ΔPaCdk5 ΔPaNip30 K-O strain (see materials and methods). PaCdk5 CDS was replaced by a hygromycin B resistance cassette while PaNip30 CDS was replaced by a nourseothricin resistance (nouR) cassette (Figure S1). While the ΔPaNip30 strain was strictly identical to the wild type, mycelium in the ΔPaCdk5 as well as in the double ΔPaCdk5 ΔPaNip30 mutant strains was slightly more pigmented and with less aerial hyphae (Figure 1). Importantly, the vegetative mutant phenotype of ΔPaCdk5 and of ΔPaCdk5 ΔPaNip30 was clearly distinguishable from the one of PaTim54 2218 . Wild-type vegetative phenotype was restored (3 [WT] out of 12 [phleoR]) when ΔPaCdk5 was co-transformed with pPaCdk5 and pBC-phleo (conferring resistance to phleomycin) together but not when transforming pBC-phleo alone (0 [WT] out of 15 [phleoR]). Complementation of ΔPaCdk5 in the ΔPaCdk5 ΔPaNip30 double mutant was achieved by crossing the ΔPaCdk5 ΔPaNip30 double mutant with a [WT] PaTim54 2218 pPaCdk5 pBCphleo transformant. Phenotypic analyses of the progeny showed that the pPaCdk5 and the pBC-phleo

  investigated genetic linkage between the pBC-hyg-PaCdk5 transgenes and the PaTim54 2218 mutation in three of the "complemented" [WT] PaTim54 2218 pBC-hyg-PaCdk5 mentioned before by crossing them with the wild type (Table 2). Strikingly, no meiotic recombination was observed between the pBC-hyg-PaCdk5 integrated transgenes and the PaTim54 2218 mutation in the three crossings. The χ 2 statistical analysis of the F1 progeny phenotypes distribution argued that for the three parental transformants analyzed, the pBC-hyg-PaCdk5 transgenes were genetically linked to the PaTim54 2218 mutation (P<10 -2 ). Similar genetical analyses revealed that in the [WT, hygR] transformants cotransformed with PaCdk5 PCR and pBC-hyg, both elements co-integrated and were genetically linked to PaTim54 2218 mutation (data not shown). Altogether, this indicated that the PaCdk5 bearing transgenes could "complement" PaTim54 2218 only when inserted in cis close to the PaTim54 2218 locus (Figure

  Strikingly, none of the PaTim54 2218 pPaCdk5 pBC-phleo progeny genotype presented a wild-type phenotype (annotated PaTim54 2218 pPaCdk5 pBC-phleo-trans in Figure 1). This demonstrated that wild-type copy of PaCdk5 did not trans-complement PaTim54 2218 although the pPaCdk5 transgenes were functional. Inversely, we wondered whether some pPaCdk5 transgenes in "non-complemented" [PaTim54 2218 ] PaTim54 2218 pPaCdk5 pBC-phleo transformants were actually functional but that they did not complement PaTim54 2218 because they were ectopically integrated. To this aim, we crossed two independent non complemented [PaTim54 2218 ] PaTim54 2218 pPaCdk5 pBC-phleo transformants to ΔPaCdk5 (Table

  PaTim54 2218 Hence, the most likely candidate gene for being altered in PaTim54 2218 remained PaTim54. Similarly as what we did for the pBC-hyg-PaCdk5 transgenes, we first tested the genetic linkage between the pBC-hyg-PaTim54 integrated transgenes and the PaTim54 2218 locus in three independent [WT] PaTim54 2218 pBC-hyg-PaTim54 complemented transformants by crossing them with the wild type. As reported in Table 5, [hygS] progeny showed [PaTim54 2218 ] mutant phenotype while all the [hygR] descendants showed wild-type phenotype. First, this confirmed the capacity of the pBC-hyg-PaTim54 transgenes analyzed to complement PaTim54 2218 . Second, χ 2 statistical analysis of the F1 progeny phenotypes distribution determined that in every transformants, the pBC-hyg-PaTim54 inserted transgene and the PaTim54 2218 mutation were genetically independent (P>0.05). This led us to conclude that PaTim54 wild-type copy did functionally trans-complement PaTim54 2218 . Altogether, these data showed that PaTim54 was the gene affected in PaTim54 2218 .

  anserina, we proceeded to mycelium fragmentation of one ΔPaTim54/ΔPaTim54 pBC-phleo-PaTim54/+ dikaryotic strain (see materials and methods). After mycelium regeneration, we recovered sixty [HygR phleoR] ΔPaTim54/ΔPaTim54 pBC-phleo-PaTim54/+ dikaryotic thalli, fourteen homokaryotic [hygR phleoR] ΔPaTim54 pBC-phleo-PaTim54 thalli but no homokaryotic [hygR phleoS]

  PaTim54 2218 as well as in PaTim54 2218 pPaCDk5 pBC-phleo-trans, two strains harboring [PaTim54 2218 ] mutant phenotype (Figure 4). On the contrary, PaTim54 gene expression was like the wild type in the other strains tested except in the complemented PaTim54 2218 pBC-phleo-PaTim54 strain, where PaTim54 expression fold-change was 1.9 compared to wild type, suggesting that overexpression of PaTim54 in this strain restored wild-type phenotype. Interestingly, PaTim54 expression was reduced to 23 % of the wild-type level in the PaTim54 2218 pBC-hyg-PaCdk5-cis transformant although this strain exhibited a wild-type phenotype. In contrast, PaTim54 expression in ΔPaCdk5, in ΔPaNip30 and in the double ΔPaCdk5 ΔPaNip30 K-O strains was not statistically different from the wild type suggesting that the gene replacements operated in these strains did not disturb PaTim54 expression. It is worthy to note that PaCdk5 gene expression was not down-regulated either in PaTim54 2218 pBCphleo-PaTim54, in PaTim54 2218 pPaCdk5 pBC-phleo-trans or in PaTim54 2218 pBC-hyg-PaCdk5-cis.

  could play a role in PaTim54 2218 -triggered HI, we constructed the PaTim54 2218 ΔPaMpk1 as well as the PaTim54 2218 PaNox1 -double mutants. Interestingly, mycelium in both double mutant strains neither looked alike PaTim54 2218 , nor ΔPaMpk1 or PaNox1 parental strains. On the contrary, mycelium in both double mutant strains was scarce (Figure 1). This observation that mycelium in both double mutant strains was more severely affected than in the single suggested a synergistic interaction between PaTim54 2218 and ΔPaMpk1 as well as between PaTim54 2218 and PaNox1 -regarding vegetative growth. With the aim to assay HI in PaTim54 2218 ΔPaMpk1 and PaTim54 2218 PaNox1 - double mutants, we challenged both strains with P. chrysogenum. We observed that cell death in P. chrysogenum at the confrontation with PaTim54 2218 , PaNox1 -, and ΔPaMpk1 single mutants was abolished as previously shown (Figure 1). The result was similar for PaTim54 2218 ΔPaMpk1 and PaTim54 2218 PaNox1 -double mutants. DAB staining was slightly decreased in PaTim54 2218 PaNox1 - double mutant while it was strongly reduced in PaTim54 2218 ΔMpk1 double mutant compared to in the wild type. Hence, ΔPaMpk1 mutation was clearly epistatic over PaTim54 2218 mutation, while PaNox1 -mutation was only partially epistatic over the PaTim54 2218 mutation. Since vegetative growth in both double mutants was similarly affected, both showing scarce and flat mycelium (Figure 1), the fact that DAB staining in PaTim54 2218 PaNox1 -when challenged with P. chrysogenum was just slightly decreased argued that drastic DAB staining loss observed in PaTim54 2218 ΔMpk1 when challenged with P. chrysogenum was unlikely due to reduced mycelium density or growth defects. This led us to conclude to a major role of the PaMpk1 MAPK, in contrast to the PaNox1 NADPH oxidase that may have a limited role, in regulating the massive oxidative burst triggered in PaTim54 2218 .

  whether the Cytochrome c respiratory pathway was disturbed in PaTim54 2218 , we quantified the expression of the PaAOX gene by RT-qPCR. Strikingly, strains harboring [PaTim54 2218 ] phenotypes, namely PaTim54 2218 and PaTim54 2218 pPaCdk5 pBc-phleo-trans, exhibited potent overexpression of the PaAOX gene, the hallmark of Cytochrome c respiratory pathway mutants (Figure 4). In particular, the PaTim54 2218 mutant showed a PaAOX gene expression fold-change of ten compared to wild type. In the other strains tested and especially in the [WT] PaTim54 2218 pBC-phleo-PaTim54 complemented strain, PaAOX gene expression was like in the wild type. This last result strongly suggested that the PaTim54 2218 mutation impaired the respiratory chain.

  PDC 2218 phenotypes result from transcriptional down-regulation of PaTim54 Following classical genetic cartography and positional cloning procedures, we characterized a 1560 bp deletion as responsible for the pleiotropic phenotypes in the PDC 2218 mutant. This deletion identified in PDC 2218 genome removes the 5' end of PaNip30, a putative gene with unknown function, together with most of the putative 3'UTR of PaCdk5, the homologue of the yeast cyclin-dependentkinase Cdk5. Several arguments immediately ruled out the possibility that PaNip30 was the gene affected in PDC 2218 . Additionally, several strong arguments, especially complementation of PDC 2218 with a wild-type copy of PaCdk5, suggested that PaCdk5 might be the gene affected in PDC 2218 . However, targeted gene deletion and cis-versus trans-complementation and functional complementation experiments supported by Mendelian genetic analyses pointed to PaTim54 as the only gene affected in PDC 2218 . Although PaCdk5 deletion points to a role of this putative cyclin in aerial hyphae production as well as mycelial pigmentation in P. anserina as well, we want to emphasize that PaTim54 fully restores wild-type phenotypes in PaTim54 2218 mutants (i.e, aerial hyphae, pigmentation, growth rate, thermosensitivity, longevity, mitochondrial defects and hyphal Interference) arguing that only PaTim54 function is altered in PaTim54 2218 mutants. We eventually show that PaTim54 is essential in P. anserina and that PaTim54 2218 , the allele present in PDC 2218 , is hypomorphic and triggers low level of expression of PaTim54. There is no evidence to explain the down-regulation in PaTim54 gene expression or the cis-suppressing effect of PaCdk5 bearing transgenes. Further investigations including genome sequencing of several cis-suppressed [WT] PaTim54 2218 pBC-hyg-Cdk5 transformants will be required to understand this cis-suppression effect of PaCdk5 bearing transgenes. However; since transformation of PaTim54 2218 with PaCdk5 PCR , a PCR fragment not overlapping the PaTim54 2218 deletion, leads to cis-suppression as well, we assume that cis-suppression by PaCdk5 bearing transgenes does not rely on the comeback of missing regulatory sequences controlling PaTim54 expression and deleted in PaTim54 2218 .

  mutant has not been straightforward and P. anserina amenability for genetic analysis greatly helped us to differentiate "false positive" complementation (of PaTim54 2218 by PaCdk5) from true trans-complementation of PaTim54 2218 by PaTim54, thus evidencing that PaTim54 was the actual gene whose decreased expression triggers the HI phenotypes. In fine, the study of the PaTim54 2218 hypomorphic mutant gave us the opportunity to get insights into the functions of PaTim54 in relation with defense and aging, although this gene is essential in P. anserina.

Figure 1 :

 1 Figures captions:

Figure 2 :

 2 Figure 2: Growth rate on M2 standard medium. A) Growth rate at 27°C, the standard temperature.

Figure 3 :

 3 Figure 3: Chromosome map of the PaTim54 2218 (PDC 2218 ) locus. Upper part, diagram of the whole

Figure 4 :

 4 Figure 4: PaCdk5, PaTim54 and PaAOX genes expression level. Expression analysis by RT-qPCR of

Figure 5 :

 5 Figure 5: Mitochondria and nucleoids in PaTim54 2218 . A) Epifluorescence microscopy photographs of

Figure 6 :

 6 Figure 6: Mitochondria of P. anserina during HI. Z series photographs of hyphae of P. anserina (P. a)

Figure 7 :

 7 Figure 7: mtDNA level quantification and senDNA analysis. A) Copy number analysis by qPCR of

  for numerical values of standard errors). B) Senescent DNA (senDNA) analysis. DNA was digested by HaeIII and stained with Ethidium Bromide. Two DNA sample replicates (1 and 2) -used for the mtDNA level quantification-per genotype were analyzed. MW, Molecular Weight marker. Bands sizes are indicated in base pairs (bp). Wtsen, genomic DNA extracted from senescent WT strain. * Senescent DNA. Senescent DNA was not detected in the DNA samples used for the qPCR-based mtDNA quantification experiment.

  Figure 2:

Table 1 : strains and genotypes

 1

	Tables:
	genotype	Detailed genotype	resistance markers	reference
	wild type	big S	-	(Rizet, 1953b)
	∆mus51	∆mus51::su8-1	-	(Lambou et al., 2008)
	∆mus51	∆mus51::phleoR	phleoR	(El-Khoury et al., 2008)
	mito-GFP	mito-GFP::hygR	hygR	(Sellem et al., 2007)
	ΔPaMpk1	ΔPaMpk1::hygR	hygR	(Kicka et al., 2006)
	PaNox1 -	IDC 343	-	(Malagnac et al., 2004)
	ΔPaTrx2	ΔPaTrx2::phleoR	phleoR	(Malagnac et al., 2007)
	PaTim54 2218	PDC 2218	-	(Haedens et al., 2005)
				This study
	ΔPaCdk5	ΔPaCdk5::hygR	hygR
	ΔPaNip30	ΔPaNip30::nouR	nouR
	ΔPaTim54*	ΔPaTim54::hygR	hygR
	ΔPaCdk5 ΔPaNip30		hygR nouR
	ΔPaCdk5 pPaCdk5 pBC-phleo		hygR phleoR
	ΔPaCdk5 ΔPaNip30 pPaCdk5 pBC-phleo		hygR phleoR nouR
	mat+/mat-ΔPaTim54/+		hygR

Table 2 : PaTim54 2218 pBC-hyg-PaCdk5 X WT

 2

	PaTim54 2218 pBC-hyg-PaCdk5 X WT	phenotypes
		[WT hygR]	[WT hygS]	[PaTim54 2218 hygS]	Genetic linkage : χ 2 test
	2	21	15	0	Yes: d<2.7cM : P<10 -2
	3	21	27	0	Yes: d<2.0cM : P<10 -9
	4	22	23	0	Yes: d<2.1cM : P<10 -4
	transformant number	PaTim54 2218 , pBC-hyg-PaCdk5	+,+	PaTim54 2218 , +
			genotypes

Table 3 : ΔPaCdk5 pPaCdk5 pBC-phleo X WT

 3

	ΔPaCdk5 {pPaCdk5 pBC phleo} X		phenotypes
	WT	[hygR phleoR]	[hygS phleoS]	[hygS phleoR]	[hygR phleoS]
	2	6 [WT]	15 [WT]	9 [WT]	12 [ΔPaCdk5]
	6	9 [WT]	12 [WT]	10 [WT]	13 [ΔPaCdk5]
	7	11 [WT]	14 [WT]	10 [WT]	13 [ΔPaCdk5]
	transformant number	ΔPaCdk5, pPaCdk5 pBC-phleo	+, +	+, pPaCdk5 pBC-phleo	ΔPaCdk5, +
			genotypes

Table 4 : ΔPaCdk5 pPaCdk5 pBC-phleo X PaTim54 2218

 4

	ΔPaCdk5 {pPaCdk5 pBC phleo} X		phenotypes
	PaTim54 2218	[hygR phleoR]	[hygS phleoS]	[hygS phleoR]	[hygR phleoS]
	2 (F1)	7 [WT]	9 [PaTim54 2218 ]	7 [PaTim54 2218 ]	17 [ΔPaCdk5]
	6 (F1)	9 [WT]	16 [PaTim54 2218 ]	8 [PaTim54 2218 ]	10 [ΔPaCdk5]
	7 (F1)	11 [WT]	8 [PaTim54 2218 ]	11 [PaTim54 2218 ]	4 [ΔPaCdk5]
	transformant number	ΔPaCdk5,	PaTim54 2218 , +	PaTim54 2218 ,	ΔPaCdk5, +
		pPaCdk5 pBC-phleo		pPaCdk5 pBC-phleo
			genotypes

Acknowledgments

We are greatly indebted to Pr. Annie Sainsard-Chanet and to Dr. Carole Sellem for their expertise in senescence in Podsopora anserina, to Sylvie Cangemi for technical help and to Charlène Girodet master student for her help. We want to thank the ImagoSeine facility, member of the France BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04, « Investmentsfit the future »).

Funding

This work was funded by Université Paris-Diderot intramural funding.

First identified in S. cerevisiae as an essential gene by [START_REF] Kerscher | The Tim54p-Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane[END_REF], other groups eventually obtained viable tim54 deleted strains [START_REF] Hwang | Tim54p connects inner membrane assembly and proteolytic pathways in the mitochondrion[END_REF][START_REF] Kovermann | Tim22, the Essential Core of the Mitochondrial Protein Insertion Complex, Forms a Voltage-Activated and Signal-Gated Channel[END_REF].

We attempted to delete PaTim54 to functionally assess PaTim54 loss-of-function. A Δmus51 strain (inactivated for the NHEJ repair system; see materials and methods), was transformed with a hygromycin B-resistance cassette designed to delete PaTim54 and we obtained 40 [hygR] transformants with no mutant vegetative phenotype. For twelve of these [hygR] transformants, integration of the deletion cassette was tested and validated by PCR (see materials and methods).

Four of these validated transformants were crossed with the wild type to obtain pure homokaryotic ΔPaTim54 (devoid of Δmus51) in the F1 progeny. In P. anserina, two kinds of ascospores can be found in asci. In a minority of asci, small homokaryotic ascospores can be recovered (along with large self-fertile dikaryotic ascospores), while the vast majority of asci only present four large mat+/matself-fertile dikaryotic ascospores. None of the four crossings yielded homokaryotic [hygR] ΔPaTim54 progeny. However, second division segregation (SDS) asci containing 4 [hygR] dikaryotic ascospores could be recovered. These asci contained self-fertile dikaryotic ascospores of the mat+/mat-ΔPaTim54/+ genotype that gave rise to wild-type growing mycelium. We then harvested homokaryotic progeny from two different self-fertile dikaryotic mat+/mat-ΔPaTim54/+ strains. For the first strain, only 18 [hygS] out of 36 ascospores germinated and for the second strain, 14 [hygS]/ 36 harvested ascospores germinated. All together, these data suggested that ΔPaTim54 deletion was lethal and/or impaired for ascospore germination, and both phenotypes being recessive.

Since homokaryotic ΔPaTim54 could not be obtained, we could not verify the strain by Southern blotting but we validated PaTim54 loss-of-function by complementation. To this purpose, we generated a pBC-phleo-PaTim54 plasmid by subcloning wild-type PaTim54 from pPaTim54 into pBCphleo. In a first step, this pBC-phleo-PaTim54 plasmid was transformed into PaTim54 2218 and we obtained 46 [WT] complemented out of 54 [phleoR] transformants. We assumed that in these complemented transformants, the pBC-phleo-PaTim54 transgenes were functional. We then transferred the pBC-phleo-PaTim54 transgenes in a ΔPaTim54 strain in two steps. First, we used the

Conflict of interest:

The authors declare that there are no conflicts of interest neither commercial affiliations.