Nicolas Valette

Julien Renou

Alexis Boutilliat

Antonio José Fernández-González

Valérie Gautier

Philippe Silar

Christophe Guyeux

Jean-Claude Charr

Stéphane Cuenot

Christophe Rose

Eric Gelhaye

Mélanie Morel-Rouhier

OSIP1 is a self-assembling DUF3129 protein

Keywords: OSIP1, fungi, cell wall, stress, oak extractives, secretome

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

Introduction

During evolution, fungi had to adapt to environmental constraints. The secretome, i.e. the proteins secreted in the extracellular medium, is a good marker of fungal physiology and trophic modes. Indeed, the secretome is involved in the first steps of the symbiosis or infection establishment and plays essential roles in plant biomass degradation [START_REF] Kämper | Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis[END_REF][START_REF] Bouws | Fungal secretomes-' lb x f white biotechnology[END_REF][START_REF] Vincent | Secretome of the free-living mycelium from the ectomycorrhizal basidiomycete Laccaria bicolor[END_REF]. Fungal secretomes are composed of degradative enzymes such as proteases, lipases, Carbohydrate-Active enZymes (CAZymes), and ligninolytic enzymes for some wood-decaying species [START_REF] Zhu | Comparative analysis of the secretomes of Schizophyllum commune and other wood-decay basidiomycetes during solidstate fermentation reveals its unique lignocellulose-degrading enzyme system[END_REF][START_REF] Pellegrin | Comparative analysis of secretomes from ectomycorrhizal fungi with an emphasis on Small-Secreted Proteins[END_REF]. This degradative system has been intensively studied due to its important application in lignocellulose biomass valorization. In plant-associated fungi, other proteins can be secreted to modulate plant immunity and establish symbiosis or allow pathogenic infection [START_REF] Plett | A secreted effector protein of Laccaria bicolor is required for symbiosis development[END_REF][START_REF] Pazzagli | Purification, characterization, and amino acid sequence of cerato-platanin, a new phytotoxic protein from Ceratocystis fimbriata f sp platani[END_REF]Frias et al., 2011;[START_REF] Baccelli | Cerato-platanin shows expansin-like activity on cellulosic materials[END_REF]. Some of them, the hydrophobins, are involved in the attachment of fungal structures to different kinds of surfaces and the development of hyphae at the water/air interface [START_REF] Wessels | Hydrophobin genes involved in formation of aerial hyphae and fruit bodies in Schizophyllum[END_REF][START_REF] Wessels | Fungal hydrophobins: proteins that function at an interface[END_REF]. In fungal pathogens, hydrophobins might act as virulence factors to enhance fungal infection [START_REF] Ruocco | Multiple roles and effects of a novel Trichoderma Hydrophobin[END_REF][START_REF] Kubicek | Purifying selection and birth-and-death evolution in the class II hydrophobin gene families of the ascomycete Trichoderma/Hypocrea[END_REF], while in symbiotic associations, these proteins could be involved in mycorrhizae formation [START_REF] Plett | Phylogenetic, genomic organization and expression analysis of hydrophobin genes in the ectomycorrhizal basidiomycete Laccaria bicolor[END_REF]. Most of the studied hydrophobins are directed to the extracellular medium through the secretory pathway. However, they often remain associated with the fungal cell wall and can be found inside fruiting bodies and on the surfaces of hyphae, spores and conidia [START_REF] Dynesen | Surface hydrophobicity of Aspergillus nidulans conidiospores and its role in pellet formation[END_REF][START_REF] Linder | Hydrophobins: Proteins that self assemble at interfaces[END_REF]. Many other secreted proteins have been identified but remain of unknown function. This is the case for SSP (for Small Secreted Proteins with sequence less than 300 amino acids) [START_REF] Alfaro | Comparative analysis of secretomes in basidiomycete fungi[END_REF]. The percentage of SSP-coding genes in the genomes of saprophytic fungi such as Phanerochaete chrysoporium, Trametes versicolor or Aspergillus fumigatus is similar to the one of the ectomycorrhizal fungus Laccaria bicolor (between 2 and 3 % of the predicted gene models) [START_REF] Pellegrin | Comparative analysis of secretomes from ectomycorrhizal fungi with an emphasis on Small-Secreted Proteins[END_REF]Valette et al., 2016). At the protein level, SSPs represent between 4 and 12% of the proteins identified in the secretomes of various Aspergillus species grown on sugar beet pulp or wheat bran (Valette et al., 2016).

However, only few have been functionally characterized. One SSP of the lignolytic fungus Pleurotus ostreatus is involved in the regulation of the lignolytic system by modulating expression and activity of aryl-alcohol oxidases, aryl-alcohol dehydrogenases and versatile peroxidases [START_REF] Feldman | A role for small secreted proteins (SSPs) in a saprophytic fungal lifestyle: Ligninolytic enzyme regulation in Pleurotus ostreatus[END_REF] and in the transition from primary to secondary metabolism, development, aging, and fruiting body initiation [START_REF] Feldman | Manipulating the expression of Small secreted protein 1 (Ssp1) alters patterns of development and metabolism in the white-rot fungus Pleurotus ostreatus[END_REF].

In a previous analysis, we have highlighted the up-regulation of various SSP-coding genes of the lignolytic fungus Phanerochaete chrysosporium in presence of oak extractives [START_REF] Thuillier | Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: Focus on a new glutathione transferase[END_REF][START_REF] Fernández-González | Oak extractive-induced stress reveals the involvement of new enzymes in the early detoxification response of Phanerochaete chrysosporium[END_REF]. Oak extractives are mainly composed of phenolic compounds and flavonoids [START_REF] Zhang | A review of polyphenolics in oak woods[END_REF][START_REF] Fernández-González | Oak extractive-induced stress reveals the involvement of new enzymes in the early detoxification response of Phanerochaete chrysosporium[END_REF].

These molecules are released from wood during the degradative process and can be toxic for cells by various mechanisms such as metal and free radical scavenging activity, direct interaction with enzymes, perturbation of ionic homeostasis and disruption of membrane and cell wall integrity (Valette et al., 2017). One of these up-regulated genes retained our attention because the corresponding protein was also detected at high amount in the secretome of another white rot fungus Trametes versicolor grown on oak wood chips (Deroy et al., unpublished). This protein was thus named OSIP1 for Oak Stress Induced Protein. It shows no sequence homology with characterized proteins in the databases. In this study we used two fungal models to decipher the role of OSIP1: Podospora anserina for functional analysis because genetic engineering is easy, contrary to P. chrysosporium or T. versicolor; and P. chrysoporium for the biochemical analysis because only PcOSIP1 was successfully produced as a recombinant protein.

Results

Comparative genomic analysis reveals that OSIP1 is widespread in fungi

OSIP1 sequences from the ascomycete P. anserina (PaOSIP1: ProtID JGI 208230, a.k.a.

Pa_5_3780 according to the P. anserina genome project) and the basidiomycete P. chrysosporium (PcOSIP1: ProtID JGI 2981896) were used as templates to search for fungal sequences using the BlastP search tool onto the whole fungal JGI database (Mycocosm from Joint Genome Institute). The sequence of P. anserina P209725 (ProtID JGI 209725) was also used as template since it displays 38% similarity with PaOSIP1). A total of 1057 protein sequences were retrieved and analyzed by clustering. These sequences grouped into nine clusters (Fig. 1A). PaOSIP1 and PcOSIP1 belonging to cluster 1, this latter was thus named OSIP1 cluster (species and accession numbers are available in Table S1). It gathers sequences from both ascomycetes (Pezizomycotina) and basidiomycetes (Agaricomycotina and Pucciniomycotina). Moreover, this analysis revealed that OSIP1 is present in genomes of wood decay fungi, and also in mycorrhizal, pathogenic fungi and other saprotrophs (Fig. 1A).

Although OSIP1 was found induced by oak extractives in lignolytic fungi [START_REF] Thuillier | Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: Focus on a new glutathione transferase[END_REF]Deroy, unpublished), this genomic analysis showing that OSIP1 is present in fungi with various trophic modes, argues against its direct involvement in the lignolytic process and suggests a more general role in fungal physiology.

OSIP1 sequences contain a DUF3129 domain, like some appressoria-specific proteins OSIP1 sequences exhibit a signal peptide of secretion, a conserved DUF3129 domain and a variable C-terminal tail (Fig. 1B). In ascomycetes, 8 cysteinyl residues are conserved, while only four have been detected in the analyzed basidiomycete sequences (Fig. 1B and Fig S1 ).

The DUF3129 domain is not restricted to OSIP1 sequences. It can be identified in both ascomycetes and basidiomycetes and was retrieved in 442 sequences from the Pfam 31.0 database. Although most of the proteins having the DUF3129 are annotated as uncharacterized proteins, some of them are described as being related to CAS1 (Colletotrichum gloeosporioides appressoria-specific protein), MAS (Magnaporthe appressoria-specific protein), gEgh16 from Blumeria graminis proteins, all being putatively involved in fungal cell wall remodeling. A phylogenetic analysis was performed with the sequences from the OSIP1 cluster and the 442 sequences containing the DUF3129 domain (Fig. 1C). PaOSIP1 clusters with ascomycete sequences but independently of CAS1 from C. gloeosporioides, MAS3 from M. grisea and gEgh16 from B. graminis. Among basidiomycetes, sequences from Agaricomycotina with lignolytic, saprotrophic or mycorrhizal lifestyles (Group A) cluster independently from sequences of pathogenic basidiomycetes from Pucciniomycotina and Agaricomycotina (Group B) (details are given in Table S1).

PaOSIP1 maintains cell wall integrity under caspofungin stress

To functionally characterize OSIP1, the OSIP1 knock-out mutant was generated in P. anserina (PaOSIP1 Δ ) (Fig S2). PaOSIP1 Δ was tested for its ability to grow on various carbon sources (cellobiose, fructose, cellulose, pectin, glucose) and various biomasses (whatman paper, hay, miscanthus, wood chips). No differences in growth, sporulation, nor appressorialike structure formation (as described in [START_REF] Brun | Functions and regulation of the Nox family in the filamentous fungus Podospora anserina: a new role in cellulose degradation[END_REF] was highlighted between the wild type and the PaOSIP1 Δ mutant strains in the tested conditions (data not shown). Because the DUF3129 domain is present in cell wall remodeling proteins, cell wall destabilizing agents were thus tested. No phenotype was observed for Congo red that prevents glucan microfibril bl l b b β-1,3 glucans [START_REF] Nodet | Morphogenetic effects of Congo red on hyphal growth and cell wall development of the fungus Saprolegnia monoka[END_REF] nor Calcofluor white, which binds chitin. By contrast, a significant deleterious growth phenotype was observed for the mutant compared to the wild type in presence of caspofungin (Fig. 2). Caspofungin is a cell wall-targeting antifungal compound extensively used in clinical settings for the treatment of infections caused by diverse fungi. Caspofungin inhibits the synthesis of β-1,3-glucan, a crucial cell wall component for many fungi, by targeting the β-1,3-glucan synthase (encoded by fks1) in a non-competitive way [START_REF] Van Den Bossche | Echinocandins -an update[END_REF][START_REF] Aguilar-Zapata | Echinocandins: The Expanding Antifungal Armamentarium[END_REF].

Functional complementation of PaOSIP1 Δ by PaOSIP1 restored the growth defect confirming the role of PaOSIP1 in protecting cell wall under caspofungin treatment.

PaOSIP1 Δ mutant is affected in extracellular matrix production

To check whether caspofungin sensitivity was due to a thinner cell wall in the mutant compared to the wild type, SEM imaging was performed after cryosection of the hyphae (Fig.

3

). The whole reconstituted images are shown as supplementary data. The thickness of the cell wall was measured (n>50) based on the microscopic images. No difference was detected between the WT and the mutant, nor between the caspofungin and the control condition. The measured cell wall thickness of hyphae cross-sections was around 150 nm for all conditions analysed. Interestingly, the main difference highlighted by comparing SEM images of both mutant and WT strains was the extracellular matrix (ECM) density. Fungal extracellular matrix is mainly composed of carbohydrates and proteins in complex interactions. This matrix is strongly reduced in PaOSIP1 Δ even in the absence of caspofungin (Fig. 3). Since this matrix was shown to prevent drugs from reaching their cellular targets [START_REF] Mitchell | Fungal Super Glue: The Biofilm Matrix and Its Composition, Assembly, and Functions[END_REF], this could explain why PaOSIP1 Δ showed an increased sensitivity to the antifungal caspofungin.

Recombinant OSIP1 self-assembles as fibers and forms a gel

The heterologous Escherichia coli system was tested for recombinant PaOSIP1 and PcOSIP1

productions. Only PcOSIP1 expressed in E. coli was successfully produced. After purification, 3 mg of pure protein per liter of bacterial culture were obtained. To check whether the protein was correctly folded, far-UV circular dichroism analysis was performed.

Spectrum of PcOSIP1 revealed secondary structures, mainly alpha helices as highlighted by a positive band at 190 nm and two negative bands at 208 and 222 nm (Fig. 4A).

After purification, PcOSIP1 rapidly self-assembled as big oligomers in Tris-NaCl buffer (30 mM Tris-HCl pH 8.0, 200 mM NaCl), as shown by dynamic light scattering (DLS) analysis (Fig. 4B). Huge hydrodynamic radii (Rh) of 789.3 ± 388 nm and 2 281 ± 880 nm were respectively measured for 56.7% and 28.7% of the PcOSIP1. This aggregation was not observed when the protein was dialyzed in 50 mM phosphate buffer pH 8.0 directly after purification, suggesting that this process was driven by the physicochemical properties of the buffer. To check whether this aggregation was due to protein instability and thus precipitation or rather a specific organization, the macromolecular structures of PcOSIP1 was analyzed by Atomic Force Microscopy (AFM). In Tris-NaCl buffer, PcOSIP1 was able to self-assemble into fibers with a mean diameter of 1.5 ± 0.2 nm (Fig. 4C). In phosphate buffer, a crown structure was evidenced. This crown organization could be the transient states of PcOSIP1 fibril formation, since such structure was already described in transient states of the human αsynuclein fibril formation that contributes to Parkinson's disease [START_REF] Lashuel | Alpha-synuclein, especially the Parkinson's disease-associated mutants, forms porelike annular and tubular protofibrils[END_REF][START_REF] Apetri | f α-Synuclein Oligomers: Characterization by Raman and Atomic Force Microscopy[END_REF].

All the experiments described above were performed directly or few days after protein purification. Interestingly, storing PcOSIP1 in Tris-NaCl buffer in the freezer led to the formation of a gel, that was quite compact and elastic (Fig. 4D). This specific feature was already described for other proteins as α-synuclein that can form gels in buffer at pH 7.4 in the presence of NaCl [START_REF] Semerdzhiev | Hydrophobic-Interaction-Induced Stiffening of Alpha-Synuclein Fibril Networks[END_REF]. These atypical properties of self-assembly and jelly structure formation support a putative structural role of OSIP1 protein within the fungal cell wall or the extracellular matrix.

Oak-extractives affect PaOSIP1 Δ growth

During wood degradation, extractives act as important stressors for fungal cells [START_REF] Thuillier | Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: Focus on a new glutathione transferase[END_REF]Valette et al., 2017 ;[START_REF] Fernández-González | Oak extractive-induced stress reveals the involvement of new enzymes in the early detoxification response of Phanerochaete chrysosporium[END_REF]. Some of them can act directly on cell wall integrity. For example, similarly to caspofungin, cinnamaldehyde and b β-1,3-glucan synthesis within the fungal cell wall [START_REF] Bang | Inhibition of fungal cell wall synthesizing enzymes by trans-cinnamaldehyde[END_REF][START_REF] Piotrowski | Plantderived antifungal agent poacic acid targets b-1,3-glucan[END_REF]. Because OSIP1 gene expression was induced by oak extractiveinduced stress both in P. chrysosporium [START_REF] Thuillier | Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: Focus on a new glutathione transferase[END_REF] and P. anserina (data not shown), this condition was used to test PaOSIP1 Δ growth phenotype. The results showed a growth delay of PaOSIP1 Δ compared to the wild type in presence of wood extractives, which was observable from 5 days (Fig. 5). This phenotype was partially restored by functional complementation with PaOSIP1. These results support the hypothesis that PaOSIP1 could participate in cell wall protection against extractives toxicity.

The deletion of OSIP1 strongly modifies the composition of the secretome and the cellwall related proteins of P. anserina in presence of oak sawdust

To analyze how the deletion of PaOSIP1 affects the fungal physiology in a context of biomass degradation, a proteomic analysis was performed for the PaOSIP1 Δ mutant grown in presence of oak sawdust, in comparison with the wild type strain. The comparative analysis of the secreted proteins reveals strong modification of the global secretome of PaOSIP1 Δ mutant compared to the wild type strain (Fig. 6A). Indeed, over the 250 proteins detected, 150 were more abundant (Protein abundance index (PAI) fold>2) or specifically identified in the mutant compared to the wild type, while this number was only 41 in the case of the wild type.

By looking at the various classes of enzymes that were more abundant in the mutant secretome compared to the WT, no clear specificity was observed except for proteases that were less represented (Fig. 6B). Globally, more than half (around 60%) of the detected proteins of a specific class were more abundant in the secretome of the mutant compared to the wild type. It is likely that the deletion of OSIP1 globally affected the secretion process of the enzymes.

Glycoside hydrolases (GH) were the most abundant proteins with a total of 66 GH detected in both secretomes. By looking at the individual GH families, we showed again that the increase in protein abundance is not restricted to specific families, but was observed for many of them (Fig. 6C). However, it is interesting to note that 16 GH families (over the total of 29) were specifically detected in the mutant strain, especially GH5, GH11 and GH43 with at least 3 isoforms.

A high number of cell wall-related proteins, in particular glucan-acting enzymes have also been found more abundant in the secretome of the mutant strain (Fig. 6D). Moreover, many

Wall Stress responsive Component (WSC) proteins have been detected. These proteins serve as sensors of external stress cues upstream of cell wall integrity (CWI) pathway in Saccharomyces cerevisiae [START_REF] Verna | A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae[END_REF] and Aspergilli [START_REF] Futagami | Putative stress sensors WscA and WscB are involved in hypo-osmotic and acidic pH stress tolerance in Aspergillus nidulans[END_REF][START_REF] Dichtl | Deciphering cell wall integrity signalling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases[END_REF]. In line with this observation, respectively 3 and 6 DUF1996-containing proteins were found more abundant and specifically detected in the mutant among the 10 detected in total (Supplementary data). These proteins are of unknown function, however the DUF1996 domain has been associated to fungal stress sensing and response (Tong et al., 2016a and2019).

Discussion

In this study, we characterized a new fungal protein, which participates in cell wall fitness under stress. The comparative genomic analysis revealed that such proteins are widely present in fungi, suggesting their involvement in a general process of stress rescue. All analyzed OSIP1 possess a domain of unknown function DUF3129 that was previously identified by few studies in proteins of both plant and insect pathogens [START_REF] Shang | Divergent and convergent evolution of fungal pathogenicity[END_REF]. DUF3129 is an expanded gene family highly expressed during infection in nematode-trapping fungi that form adhesive branches and adhesive knobs [START_REF] Andersson | Interspecific and host-related gene expression patterns in nematode-trapping fungi[END_REF]. The role of this domain was attributed to the cell wall remodeling for fungal penetration to host cuticules with an unclear mechanism [START_REF] Justesen | Isolation and characterization of two novel genes expressed in germinating conidia of the obligate biotroph Erysiphe graminis f. sp. hordei[END_REF][START_REF] Xue | Two novel fungal virulence genes specifically expressed in appressoria of the rice blast fungus[END_REF][START_REF] Grell | A Blumeria graminis gene family encoding proteins with a C-terminal variable region with homologues in pathogenic fungi[END_REF][START_REF] Cao | The Magas1 gene is involved in pathogenesis by affecting penetration in Metarhizium acridum[END_REF].

More recently, seven DUF3129 proteins of the insect pathogenic fungus Metarhizium robertsii were found localized to cellular lipid droplets mediating their degradation and subsequently controlling appressorial turgor required for infection [START_REF] Huang | Unveiling the function and regulation control of the DUF3129 family proteins in fungal infection of hosts[END_REF].

However, nothing has been described concerning the role of these DUF3129-containing proteins in saprophytic fungi. The jelly structure of the recombinant PcOSIP1 and the reduction of the extracellular polysaccharide network observed for the PaOSIP1 mutant strongly suggest the involvement of OSIP1 in the formation of such adhesive structures in fungi. The weakened extracellular matrix of the mutant could thus be responsible for the higher susceptibility of the fungus to both caspofungin and oak extractives.

This phenomenom has been already described in A. fumigatus, where the downregulation of a hydrophobin gene by a polyphenolic compound resulted in a weakened extracellular matrix and therefore increased the susceptibility of the fungi to antifungal drugs [START_REF] Luo | 3,5-Dicaffeoylquinic Acid Disperses Aspergillus Fumigatus Biofilm and Enhances Fungicidal Efficacy of Voriconazole and Amphotericin B[END_REF].

Hydrophobins are small (100-120 aa) secreted proteins characterized by the presence of eight highly conserved cysteine residues and the ability to self-assemble as amyloid-like structure and forms rodlets [START_REF] Ball | Hydrophobin Rodlets on the Fungal Cell Wall[END_REF]. Amyloids serve diverse purposes for structure, adhesion and defence in microorganisms [START_REF] Shanmugam | Microbial functional amyloids serve diverse purposes for structure, adhesion and defence[END_REF] and can be evidenced in vitro using fluorescent tool as thioflavin T, which binds to the beta sheet-rich structure characteristic of amyloid-like structure [START_REF] Groenning | Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils-current status[END_REF]. PcOSIP1 does not assemble under amyloid structure since no thioflavin T fluorescence signal was detected in any of the conditions of temperatures and buffers tested (data not shown). We have shown that PcOSIP1 was rich αhelices, while amyloid ll b β-sheets. Moreover, AFM revealed that it did not form rodlets. These experimental data, coupled to sequence analysis, allowed us to confirm that OSIP1 is a new self-assembling protein, that does not belong to the well-known class of hydrophobins.

In the context of lignocellulosic biomass degradation, the structural property of OSIP1 may be the key point explaining the way by which it participates in fungal stress resistance by protecting the cell wall. In accordance, the comparative secretome analysis of P. anserina grown on oak sawdust revealed that the PaOSIP1  mutant highly expresses WSC proteins compared to the wild type in this condition. WSC proteins are localized to the cell wall and the plasma membrane and act as sensors upstream of the cell-wall integrity pathway. In particular, WSC-1 may function in regulating cell wall biogenesis through the MAK-1 pathway in Neurospora crassa [START_REF] Maddi | WSC-1 and HAM-7 are MAPK-1 MAP kinase pathway sensors required for cell wall integrity and hyphal fusion in Neurospora crassa[END_REF]. Single deletions of the five wsc genes of Beauveria bassiana resulted in significant, but differential, increases in cellular sensitivity to cell wall perturbation, oxidation, high osmolarity, and metal ions (Tong et al., 2016b). In Aspergillus fumigatus, deletions of wsc1 caused an increased in sensitivity to caspofungin but no change in cellular sensitivity to other cell wall perturbation, alkaline pH and high temperature [START_REF] Dichtl | Deciphering cell wall integrity signalling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases[END_REF]. In the nematode-trapping fungus Monacrosporium haptotylum, a gene cluster of 5 secreted proteins that are adjacent in the M. haptotylum genome (cluster 74) is highly (>10-fold) upregulated during infection [START_REF] Andersson | Proteome of the nematode-trapping cells of the fungus Monacrosporium haptotylum[END_REF]. This cluster gathers two genes coding for WSC proteins, one gene containing the DUF3129 domain and two SSP-coding genes [START_REF] Meerupati | Genomic mechanisms accounting for the adaptation to parasitism in nematode-trapping fungi[END_REF]. This suggests a functional link between these proteins. Additionally to WSC proteins, the PaOSIP1  mutant highly expresses DUF1996-containing proteins. In B. bassiana, DUF1996-containing proteins localize in vacuoles and play significant roles in the response to cell-wall perturbation, high osmolarity, oxidation, fungicidal and multiple metal stress (Tong et al., 2016a). The absence of OSIP1 in P. anserina grown in presence of oak sawdust strongly affected the whole secretome of the fungus, likely because of the cell wall weakness. Indeed, the functionality of the cell wall integrity (CWI) and secretory systems are connected and coordinately respond to exogenous stresses through the modulation of the cell periphery and secretion [START_REF] Malavazi | The importance of connections between the cell wall integrity pathway and the unfolded protein response in filamentous fungi[END_REF].

Taking together, these data strongly suggest that OSIP1 proteins prevent cell wall stress.

Consequently, its absence affects the cell wall associated signaling pathway, leading to a deregulation of the secretion process. These data could be of great interest for both the improvement of protein secretion particularly in a context of lignocellulosic biomass degradation, and the limitation of fungal pathogenicity, for which the fungal cell wall has a crucial role [START_REF] Gow | The Fungal Cell Wall: Structure, Biosynthesis, and Function[END_REF].

Experimental procedures

Growth conditions

The P. anserina strain used in this study was derived from the S strain [START_REF] Rizet | Les phénomènes de barrage chez Podospora anserina. I. Analyse génétique des barrages entre souches S and s[END_REF][START_REF] Boucher | Species delimitation in the Podospora anserina/P. pauciseta/P. comata species complex (Sordariales)[END_REF]. Standard culture conditions, media compositions and genetic methods for this fungus have already been described [START_REF] Rizet | Sur l'analyse génétique des asques du Podospora anserina[END_REF][START_REF] Silar | Podospora anserina: from laboratory to biotechnology[END_REF] and are available at https://podospora.i2bc.paris-saclay.fr. Growth kinetics of the wild type and PaOSIP1 ∆ strains were done in M2-Agar medium and M2-Agar medium supplemented with caspofungin (500 ng/ml) and oak extractives (2 mg/ml) for 10 days at 27 °C. Oak (Quercus petraea) acetonic extract preparation has been performed as described previously [START_REF] Fernández-González | Oak extractive-induced stress reveals the involvement of new enzymes in the early detoxification response of Phanerochaete chrysosporium[END_REF].

Analysis of OSIP1 sequences

OSIP1 sequences were searched within all fungal genomes available in the Joint Genome Institute database (Mycocosm https://genome.jgi.doe.gov/programs/fungi/index.jsf) using

BlastP with a cut off of Evalue=10 -5 . Sequences of OSIP1 of Podospora anserina ((PaOSIP1 (208230 JGI) and Phanerochaete chrysosporium (ProtID 2981896 JGI) have been used as templates. Another sequence close to PaOSIP1 was added as a template (P209725 (ProtID 209725 JGI)). Evolutionary analyses were conducted in MEGA7 using the Neighbor-Joining method [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[END_REF]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The analysis involved 628 amino acid sequences.

All ambiguous positions were removed for each sequence pair. There were a total of 467 positions in the final dataset. To carry out the clustering, the Laplacian eigenmap technique [START_REF] Belkin | Laplacian eigenmaps for dimensionality reduction and data representation[END_REF] was applied with Gaussian mixture model [START_REF] Reynolds | Gaussian mixture models[END_REF] as follows. After sorting the sequences in alphabetical order, the similarity of each sequence pair were obtained from the score provided during a pairwise sequence alignment using the Needleman-Wunsch [START_REF] Needleman | A general method applicable to the search for similarities in the amino acid sequence of two proteins[END_REF] dynamic programming algorithm from Biopython [START_REF] Cock | Biopython: freely available Python tools for computational molecular biology and bioinformatics[END_REF] module (pairwise2 function). Default values for gap open and extend penalties were chosen with blosum62 matrix for amino acid substitution, leading to a matrix M of integers. A similarity matrix S has been deduced by dividing each row by its maximum, and by computing the identity matrix minus this one. The normalized Laplacian associated with the similarity matrix has been computed as follows: L = D -1 (D-S), where D is the diagonal matrix whose element in position (i,i) is the sum of the i-th row in S. Eigenvalues of L have then been computed and sorted in ascending order thanks to the numpy library [START_REF] Oliphant | A guide to NumPy[END_REF], and the N-th first eigenvalues have only be considered, where N is such that the increase between the N-th and N+1-th eigenvalue is lower than 1%. Associated and phleomycin-sensitive descendant was selected as the PaOSIP1::hygroRstrain or PaOSIP1 ∆ . Its genotype was confirmed by Southern blot analyses using digoxigenin labeled probes (Fig. S2). For functional complementation tests, PaOSIP1 coding sequence was cloned into pAKS-Genet R vector and expressed in PaOSIP1 ∆ . The presence of the gene was checked by PCR and three transformants were selected for functional complementation tests. They all restored the mutant phenotype, thus, only the results for one of them are presented.

SEM imaging of PaOSIP1  hyphal network

Cloning of PcOSIP1

Phanerochaete chrysosporium mycelium was harvested from liquid cultures in TK medium supplemented with oak extractives as previously described [START_REF] Thuillier | Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: Focus on a new glutathione transferase[END_REF]. Total RNA was extracted and purified using the RNeasy plant minikit (Qiagen) according to the nm with a bandwidth of 1 nm using a Chirascan Plus spectropolarimeter (Applied Photophysics, Ltd, UK). The mean residue ellipticity [θ]MR was calculated using Pro-Data Viewer (Applied Photophysics, Ltd, UK) software and expressed in deg. cm 2 .dmol -1 per residue.

Dynamic Light Scattering (DLS)

The homogeneity of solutions, the aggregation state and particle sizes were analyzed by granulometry on a Zetasizer Nano-S model (Malvern Instruments, Malvern, UK). The protein solution was analyzed by DLS at a final concentration of 4mg/ml either in 50 mM phosphate buffer pH8.0 or 30 mM Tris-HCl, 200 mM NaCl buffer. The supernatant of each sample was gently transferred into a quartz cuvette of 12 µl and the particle size measurements were performed in triplicate at 37°C, with alight diffusion at 173°. The data were collected in automatic mode and analyzed using the associated software DTS version 4.2 (Malvern Instruments).

Atomic Force Microscopy (AFM)

PcOSIP1, either in 50 mM phosphate buffer (pH 8.0) or 30 mM Tris-HCl and NaCl 200 mM buffer (pH 8.0), was analyzed at a starting protein concentration of 6 mg/ml. The protein solutions were carefully dialyzed to remove NaCl and diluted 10 times just prior to AFM observations. A glass coverslip was cleaned with a piranha treatment and washed in ultrapure water, before being dried in a stream of nitrogen gas. A tiny droplet of each diluted protein solution was deposited onto the glass coverslip heated at 20°C to promote a rapid drying (within 2 minutes) while avoiding the formation of concentration gradients on the substrate [START_REF] Zykwinska | Spontaneous selfassembly of SC3 hydrophobins into nanorods in aqueous solution[END_REF]. The sample was then immediately imaged by AFM. A NanoWizard® Atomic Force Microscope (JPK, Germany) operating in intermittent contact ionization. Peptide attribution and protein annotation were performed by comparing mass spectrometry data to predicted proteins in the genomes of P. anserina as well as an internal contaminant database, using X!Tandem Cyclone software (X!Tandem, Jouy-en-Josas, France). The protein annotation was completed manually by BlastP using both the NCBI (https://blast.ncbi.nlm.nih.gov) and JGI Mycocosm (https://mycocosm.jgi.doe.gov/Podan3/Podan3.home.html) databases. Blumeria graminis, Cg: Colletotrichum gloeosporioides. Because some of DUF3129containing proteins are annotated as GAS-like proteins in the pfam database, GAS1

(Glycolipid Anchored Surface) from Saccharomyces cerevisiae that has been well studied [START_REF] Ragni | The Gas family of proteins of Saccharomyces cerevisiae: characterization and evolutionary analysis[END_REF] and the GAS1 sequence of P. chrysosporium, were added to the analysis.

  with 2 M LiCl. RNAs were reverse transcribed using the masterscript kit (5 prime) following the manufacturer's protocol. The PCR reactions to amplify PcOSIP1 (Prot ID 2981896 in the Joint Genome Institute database v2.2 (previously identified as Prot ID 4474 in v2.0 of P. chrysosporium genome annotation)), have been performed with Herculase Taq (Agilent technologies) for cloning into the pEt26b (Novagen) vector for His-tagged protein production in Escherichia coli. The sequence was amplified without the

Fig. 1 :

 1 Fig. 1: Comparative genomics of fungal OSIP1

Fig. 2 :

 2 Fig. 2: Growth phenotype of PaOSIP1  in presence of caspofungin.

Fig. 3 :

 3 Fig. 3: SEM images of PaOSIP1  and WT hyphal network.

Fig. 4 :

 4 Fig. 4: Self-assembly of PcOSIP1.

Fig S1 :

 S1 Fig S1: Sequence alignment of basidiomycete OSIP1 sequences from P. chrysosporium,

Fig. S2 :

 S2 Fig. S2: Strategy for PaOSIP1 deletion. (A) Gene replacement strategy for deleting

Figure 1 Figure 6

 16 Figure 1

Acknowledgements

This work was supported by a grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (ANR-11-LABX-0002-01, Lab of Excellence ARBRE) and the Region Lorraine Research Council. We thank Thomas Bacchetta for experimental help, Sylvie Cangemi for expert technical assistance, Christophe Rose (UMR SILVA-SILVATECH INRAE) for microscopy and Alexandre Kriznik for CD data (UMR7365 UL/CNRS IMoPA, Nancy, France). Proteomics analyses were performed on the PAPPSO platform (http://pappso.inra.fr) which is supported by INRA (http://www.inra.fr), the Ile-de-France regional council (https://www.iledefrance.fr/education-recherche ), IBiSA ( https://www.ibisa.net ) and CNRS ( http://www.cnrs.fr ).

The authors declare no competing interests.

eigenvectors have then been clusterized according to a Gaussian mixture model [START_REF] Reynolds | Gaussian mixture models[END_REF], and the model selection (number of Gaussians) has been performed according to the Bayesian Information Criterion (BIC, [START_REF] Schwarz | Estimating the dimension of a model[END_REF]). To sum up, Laplacian eigenmap allowed us to map the similarity matrix in a low dimensional space of points, each point being associated to one amino acid sequence. This cloud points has been considered as the superposition of a given number of gaussian trends (the clusters), this number being determined thanks to the BIC criterion of parcimony. For further information about this sequence clustering technique, see, e.g. [START_REF] Bruneau | A clustering package for nucleotide sequences using Laplacian Eigenmaps and Gaussian Mixture Model[END_REF]. NaCl. The purified protein was finally concentrated and analyzed on 15% SDS-PAGE gel to check the purity. The concentration of the protein was determined by BC assay (interchim).

Deletion of PaOSIP1 in

Circular dichroism (CD)

Due to the incompatibility of Tris buffer, which absorbs between 180 and 260 nm, the PcOSIP1 spectrum was recorded exclusively in phosphate buffer. Directly after purification, PcOSIP1 was dialyzed in 50 mM phosphate buffer pH 8.0 using dialysis membrane (Spectra/Por, MWCO 6-8 000). Circular Dichroism spectra of PcOSIP1 was obtained in 50 mM phosphate buffer pH 8.0 at 25 °C in a quartz cuvette (1-mm path length) from 180 to 260 mode under ambient conditions was used to image the protein solutions deposited onto the glass coverslip. A standard rectangular cantilever (Nanosensors NCL-W) was employed for imaging (scan rate of 0.5 Hz), with a free resonance frequency of 174 kHz and a curvature radius of the tip of 10 nm. In order to check the reproducibility of the observed morphology, all samples were scanned at least on three different zones. Each sample was investigated using fresh tips previously cleaned by UV-ozone treatment. The height measurements were done using JPK Data Processing software (JPK, Germany).

LC-MS/MS protein identification

P. anserina wild type and PaOSIP1 ∆ strains were cultivated in flasks containing 1 g of oak sawdust and 10 ml of M2 medium without any carbon source for 1 month at 25°C. For each strain, three independent cultures were pooled before protein extraction. Proteins from the whole sample (sawdust containing mycelium and secretome) were extracted with 10 ml of 50 mM sodium acetate pH4.5 buffer for 1.5 h under shaking at 4°C. The sample was centrifuged, concentrated with centricon filter membrane (5 kDa) until around 3 ml and precipitated with cold acetone (80%). 10 µg of proteins was loaded on 12% SDS-PAGE gel. After a short migration (0.5 cm) in the stacking gel, the gels were stained with Coomassie blue and each electrophoresis track was cut into two 2-mm-wide strips. Proteomic identification was performed at the Plate-f 'A l P q P -Ouest (PAPPSO, INRA, Jouy-en-Josas, France; http://pappso.inra.fr/), according to a protocol described in [START_REF] Navarro | Automated assay for screening the enzymatic release of reducing sugars from micronized biomass[END_REF]. Briefly, the digestion of the proteins contained in the gel strips was carried out according to a standard trypsinolysis process, using modified trypsin (Promega, Charbonnières-les-Bains, France). Peptide analysis was performed by Ultimate 3000

RSLCnano liquid chromatography (Thermo Fisher Scientific, Waltham, Massachusetts, USA) coupled to a Q-exactive mass spectrometer (Thermo Fisher Scientific) using electrospray Growth kinetics of the wild type, PaOSIP1  mutant and a complemented strain (PaOSIP1  _PaOSIP1) in M2 medium supplemented with oak extractives (2 mg/ml) (n=3). The pictures show fungal growth after 10 days. S2.