The GUN mutants: new weapons to unravel ascospore germination regulation in the model fungus Podospora anserina

Abstract

In Podospora anserina as in many other ascomycetes, ascospore germination is a regulated process that requires breaking of dormancy. Despite its importance in survival and dispersal, ascospore germination in filamentous fungi has been poorly investigated and little is known about its regulation and genetic control. We have designed a positive genetic screen that led to the isolation of mutants showing uncontrolled germination, the GUN mutants. In this paper, we report on the characterization of GUN1^SG. We show that GUN1^SG is mutated in Pa_6_1340, the ortholog of Magnaporthe oryzae Pth2, which encodes a Carnitine-acetyltransferase (CAT) involved in the shuttling of acetyl-CoA between peroxisomes and mitochondria and which is required for appressorium-development. Bioinformatic analysis revealed that the mutated residue (I441) is highly conserved among the Fungi, and that the mutation has a deleterious impact on the protein function. We show that GUN1 is essential for ascospore germination and that the protein is localized both in mitochondria and in peroxisomes. Finally, epistasis studies allowed us to place GUN1 upstream of the PaMpk2 MAPK pathway and the PaNox2/PaPls1 complex in the regulation of ascospore germination. The identification of GUN1, the ortholog of Pth2, in ascospore germination, strengthens the idea of a common genetic regulation governing both appressorium development and melanized ascospore germination. In addition, we characterize the second CAT encoded in P. anserina genome, Pa_3_7660/GUP1, and we show that the function of both CATs is conserved in P. anserina.

Introduction

Fungi are eukaryotic microorganisms able to resist adverse environments and disseminate through the formation of asexual (mitospores) or sexual spores (meiospores). Representing the final product of sexual reproduction, meiospores ensure survival, dissemination and carry genetic diversity, promoting adaptation in changing environments (Hoekstra, 2005). Spore germination is a crucial step in fungal life cycle, and its successful completion is thus essential for survival and spread. Conidial germination represents the starting point of infection for important pathogenic species such as Aspergillus fumigatus or Magnaporthe oryzae, and its regulation has therefore been widely studied (Baltussen et al., 2020; Osherov and May, 2001). By contrast, little is known about the regulation of ascospore germination, although ascospores are the key dispersal propagule for many other important pathogenic species such as Leptosphaeria maculans, the main pathogen for oilseed rape cultures (Daverdin et al., 2012; Howlett et al., 2001). Although conidial germination and ascospore germination share some morphological similarities, several studies have demonstrated that their regulation is different (Trapero-Casas and Kaiser, 2007). Podospora anserina, a saprotrophic coprophilous ascomycete from the sordariales order, emerges as an excellent genetic model system to study ascospore germination. P. anserina produces asexual spermatia that are unable to germinate, as well as ascospores that represent the only germinating spores in this fungus. This species forms after fertilization pear-shaped fruiting bodies called perithecia, each harboring a few hundred to a thousand ascospores, generally embedded in groups of four inside asci (Silar, 2020). The melanized ascospores of P. anserina are in a dormant state and require a stimulus in order to germinate. P. anserina is a coprophilous fungus reported to grow preferentially on herbivores’ dung. Indeed, in the wild, the breaking of dormancy of P. anserina ascospores is usually triggered by the passage through herbivores digestive tract, leading to germination on dung. This stimulus is reproduced in laboratory conditions, by placing the ascospores on a germination medium (Silar, 2020). In P. anserina, ascospore germination takes place in several steps: i) the activation by the stimulus (dormancy breaking), ii) formation of the germination pore at the apex of the spore (opposite to the primary appendage), iii) extrusion of a germination peg/bubble from which one or several (often two) germinating hyphae emerge.

It was shown that both H₂O₂ and O₂^- Reactive Oxygen Species (ROS) are produced during ascospore germination in P. anserina, suggesting that ROS play an important role at this stage (Malagnac et al., 2004). Knock-out of the superoxide (O₂⁻) producing enzyme Nicotinamide-Adenine-Dinucleotide-Phosphate (NADPH) oxidase PaNox2 encoding gene, of the PaPls1 tetraspanin encoding gene as well as the regulatory subunit PaNoxR, leads to an almost complete abolishment of ascospore germination (Brun et al., 2009; Lambou et al., 2008; Malagnac et al., 2004). Remarkably, mutations (or deletions) of the orthologs of PaNox2 and PaNoxR genes lead to loss-of-ascospore germination ability, in both model species Sordaria macrospora and Neurospora crassa, highlighting the conservation of Nox2/B, and NoxR functions in the regulation of ascospore germination (Cano-Dominguez et al., 2008; Dirschnabel et al., 2014). Strikingly, the NADPH oxidase complexes Nox1/A, Nox2/B, NoxR and Pls1 are required for appressorium functioning in the plant pathogenic fungus M. oryzae (Lambou et al., 2008; Ryder et al., 2013, 2019). Considering that the ascospores in P. anserina and the appressorium in M. oryzae are both melanized structures, we have hypothesized that, similar components and in particular the Nox2/B-Pls1-NoxR complex regulate cellular processes such as the formation of the pore through which arise the penetration peg in M. oryzae and the germination peg in P. anserina (Brun and Silar, 2010; Malagnac et al., 2008).

The same hypothesis applies to the Mitogen-Activated-Protein-Kinase (MAPK) pathway PaMpk2/MoPmk1/ScFus3 involved in appressorium development in M. oryzae as well as in ascospore germination in P. anserina (Lalucque et al., 2012; Widmann et al., 1999; Xu et al., 1998). We have shown that loss-of-function of each of the three kinases of the MAPK cascade in P. anserina, i.e., PaTlk2, PaMkk2 and PaMpk2, abolishes ascospore germination. Conversely, the activation of the pathway in the constitutively active PaMkk2^c mutant, leads to spontaneous germination of ascospores (Lalucque et al., 2012). In N. crassa, mutants of MAK-2, the ortholog of Fus3/PaMpk2 and mutants of pp-1/Ste12, the downstream transcription factor of the cascade, show almost complete lack of ascospore germination (Li et al., 2005; Pandey et al., 2004). In addition, Knock-Out of the Ste12 ortholog in S. macrospora also impairs ascospore germination (Nolting and Pöggeler, 2006). Interestingly, these three species harbor melanized ascospores that require a stimulus to germinate and this raises questions as to the role of this pigment during spore germination.

As in other fungi, melanin in P. anserina is synthesized through an enzymatic cascade, starting with the PaPks1 Polyketide Synthase. Inactivation of this key enzyme results in total inability to produce melanin in hyphae, perithecia and ascospores, as observed in the PaPks1¹⁹³ and the ΔPaPks1 null mutants (Coppin and Silar, 2007; Gautier et al., 2021; Langfelder et al., 2003). Remarkably, non-melanized ascospores in the PaPks1¹⁹³ mutant escape from dormancy and germinate spontaneously. Since the presence of melanin in cell wall contributes to its rigidity (Gómez and Nosanchuk, 2003), it is assumed that lack of melanin weakens the ascospore cell wall, leading to “accidental”, uncontrolled germination. In line with this, culturing and crossing P. anserina on medium containing the fungicide Tricyclazole, an inhibitor of melanin production in fungi, allows ascospores bearing mutations in genes essential for germination such as PaNox2, PaNoxR, PaPls1 or PaMpk2 to spontaneously germinate (Brun et al., 2009; Coppin and Silar, 2007; Lalucque et al., 2012; Lambou et al., 2008; Malagnac et al., 2004). It is worth noting that these non-melanized ascospores are fragile and lose viability if manipulated (when moved with a needle for harvesting for instance).

A defect in ascospore melanization and germination is also observed in mutants of peroxisome biogenesis, peroxisomal β-oxidation and mitochondrial β-oxidation. In these mutants, depletion in acetyl-Coenzyme A (acetyl-CoA) the direct product of β-oxidation and the main precursor for melanin biosynthesis is supposed to account for both defects (Berteaux-Lecellier et al., 1995; Boisnard et al., 2009; Bonnet et al., 2006; Peraza-Reyes et al., 2008). Peroxisomes are organelles of fundamental importance in particular for fungal pathogenesis (Asakura et al., 2006; Imazaki et al., 2010; Peraza-Reyes et al., 2011). In M. oryzae, mutants affected in peroxisomal β-oxidation and acetyl-CoA metabolism show: defects in appressorium development, appressorium demelanization and lack of pathogenicity (Chen et al., 2016; Kretschmer et al., 2012; Wang et al., 2007). Besides, the Carnitine-acetyl transferase (CAT) Pth2 mutant was isolated in a forward genetic screen designed to uncover pathogenicity mutants in M. oryzae, underlining the central role of acetyl-CoA metabolism during M. oryzae infection (Bhambra et al., 2006; Ramos-Pamplona and Naqvi, 2006; Sweigard et al., 1998). Despite the lack of knowledge on ascospore germination, this process has never been subjected to any dedicated genetic screening. In this paper, we describe the first genetic screening of ascospore germination mutants. Previous observations have shown that a negative genetic screening of mutants defective for ascospore germination i) is time-consuming compared to positive screening, ii) may lead to the isolation of mutants nonspecifically affected in any kind of cellular processes eventually leading to lack of viability/germination of ascospores and iii) brings intrinsic issues in genetic analysis since these non-germinating mutants are blocked in their sexual/life cycle. With that in mind, we have designed a direct genetic screen aiming at isolating spontaneous germination mutants in the wild-type P. anserina strain normally showing controlled germination. Moreover, with the aim to isolate mutants of the PaNox2-PaPls1 pathway, we have also screened for suppressors of ΔPaNox2 and ΔPaPls1, for which germination was restored. In this paper, we describe the Germination UNcontrolled One (GUN1^SG) mutant, we identify and we characterize the gene affected as well as its paralogue the “GUN1 Paralogue One” gene (GUP1). The functional analysis of both genes show that GUN1 codes for the ortholog of the M. oryzae Pth2/Crat1 CAT (CAT2 in Saccharomyces cerevisiae; AcuJ in Aspergillus nidulans), GUP1 codes for the ortholog of the FacC CAT in A. nidulans and Crat2 in M. oryzae (Bhambra et al., 2006; Hynes et al., 2011; Ramos-Pamplona and Naqvi, 2006; van Roermund et al., 1999) and only GUN1 is important for ascospore germination and melanization.

Results

Isolation of Germination Uncontrolled-GUN mutants

In P. anserina, wild-type ascospores are dormant and do not germinate on standard M2 medium. They require a stimulus to germinate (see movie S1 for wild-type germination), provided in laboratory conditions in the specific germination G medium, supplemented with yeast extract (YE) to increase germination rate (Silar, 2013). Rather than screening for mutants unable to germinate, we set up a positive genetic screen allowing isolation of mutants producing spontaneously germinating ascospores on M2 medium. In parallel, we screened for suppressors of the germination defect of the ΔPaNox2 and ΔPaPls1 mutant strains (Malagnac et al., 2004, 2008) using the same protocol. Auto-fertile (mat+/mat-) mycelia of the S, ΔPaNox2 and ΔPaPls1 strains were exposed to UV mutagenesis and mutants producing spontaneously germinating ascospores on standard M2 medium were isolated (see Figure S1 and Mat. & Met.). Mutant screening allowed recovery of 22 suppressors of ΔPaNox2, 16 suppressors of ΔPaPls1, and 19 mutants from the S strain, all of them producing ascospores spontaneously germinating on standard M2 medium (Table S1). Genetic analysis of these mutants revealed that for all of them, the mutant phenotype was controlled by a single locus. We then checked the germination process in these 57 mutants by microscopic analysis. For all the suppressors of ΔPaNox2 and ΔPaPls1, and 13 mutants of the S strain, ascospore germination was morphologically abnormal. In these mutants, we could observe germination through the primary appendage. This latter selection was important to possibly discard structural mutants in which the ascospore cell wall was impaired, leading to “accidental” germination or mutants in which the cell constituting the primary appendage failed to degenerate. Indeed, in P. anserina the cell constituting the primary appendage degenerates, otherwise, a germ tube arises from this cell, leading to spontaneous germination. Only 6 of the mutants isolated from the wild-type S strain showed morphologically “normal” germination proceeding through the germination pore (Table S1 & movie S1). We speculated that these six mutants represent mutants of genes involved in the signalling pathway that controls germination and were named GUNx^SG for Germination Uncontrolledx^{Spontaneous Germination} where x stands for the mutant number. Among these mutants, one had the particular characteristic to germinate on agar plates devoid of carbon and nitrate sources while the five others did not (data not shown). We therefore started the characterization of this mutant that we named GUN1^SG. This mutant differentiated ascospores with normal shape (Figure 1A), visually normal melanization and spontaneous germination through the germination pore (Figure 1B). We determined the germination rate of this mutant on M2 medium, as well as on G+YE medium, and compared it to the wild-type. Throughout our experiments, we never observed germination of wild-type ascospores when sown onto M2 medium. In contrast, when GUN1^SG/GUN1^SG heterokaryotic ascospores were transferred onto M2 medium to estimate germination rate, 54/100 germinated. In the same experiment, 94/100 germinated on G+YE, a comparable rate to WT ascospores (92/100). Once germination was initiated, development of the mycelium produced by the GUN1^SG mutant was identical to the wild-type: the GUN1^SG mutant showed wild-type vegetative phenotype, growth rate, fertility, ascospore production, appressorium development and cellophane breaching (Figure 2, S2 & Table 2).

Figure 1: Heterokaryotic ascospores morphology & germination.

A) Ascospore morphology. (WT) GUN1/GUN1, GUN1^SG/GUN1^SG and, ΔGUN1/ΔGUN1 pGUN1/pGUN1 ascospores are fully melanized compared to ΔGUN1/ΔGUN1 ascospores which are partially demelanized: Last row shows FDS ascus composed of 2 [WT] GUN1/GUN1 and two [ΔGUN1] ΔGUN1/ΔGUN1 ascospores. Scale bar 30 μm. B) Ascospore germination. (WT) GUN1/GUN1 ascospores require G+YE medium to germinate while GUN1^SG/GUN1^SG and PaPks1¹⁹³/PaPks1¹⁹³ ascospores germinate in M2 medium. Germination occurs through the germination pore located at the tip of the ascospore. 3^rd row, the melanized ascorpore is of the PaPks1/PaPks1 genotype and the non-melanized ascospore is of the PaPks1¹⁹³/PaPks1¹⁹³ genotype. Scale bar 10μm.

Figure 2: Growth on different carbon sources.

Pictures were taken after 4 days of culture. All the media have same composition except for the carbon source. The Tween 40 control medium and the oleic acid medium contain 0,5% Tween 40. Scale bar 1 cm.

Table 1: Strains list

Table 2: Cellophane penetration assay.

The listed strains were cultured for 5 days on M2 medium topped with a cellophane layer at 27 °C. After 2-, 3-, 4- and 5-days of culture, the cellophane layer was removed to check fungal growth in the medium underneath. The day the cellophane was breached is indicated. 3 replicates were made for each day of culture. This experiment was repeated twice.

GUN1 encodes a Carnitin-acetyltransferase (CAT)

The gene mutated in GUN1^SG was identified through whole-genome sequencing. To that end, this mutant was backcrossed five times with the wild-type strain beforehand in order to eliminate most of the mutations generated during UV mutagenesis but not genetically linked to the mutation responsible for the mutant phenotype. The analysis of GUN1^SG whole-genome sequence revealed the presence of six silent mutations, and three missense mutations, one in Pa_1_13700, which encodes a putative protein of unknown function, another in Pa_5_7800, encoding a putative phosphoketolase and the last one in the Pa_6_1340 CDS, where an isoleucine was changed into an asparagine (I441N), caught our attention (Figure 3). RNA-seq data indicated that Pa_6_1340 was strongly induced (Fold Change = 56) during ascospore germination, emphasizing the involvement of this gene during ascospore germination (Demoor, unpublished data). This CDS encodes a putative peroxisomal/mitochondrial Carnitine-acetyltransferase (CAT) of 643 amino acids (Masterson and Wood, 2000; Seccombe and Hahn, 1980; Strijbis and Distel, 2010; Strijbis et al., 2010). Homologs of this gene have previously been studied in S. cerevisiae, Aspergillus nidulans, Giberella zeae, Sclerotinia sclerotiorum and M. oryzae where they are involved in acetate/acetyl-CoA metabolism and more particularly in pathogenicity and appressorium development in the phytopathogenic species mentioned (Bhambra et al., 2006; Hynes et al., 2011; Liberti et al., 2013; Ramos-Pamplona and Naqvi, 2006; Son et al., 2012). Regarding the role of acetate and peroxisomes in the control of germination in P. anserina, and given the induction of Pa_6_1340 during ascospore germination, this gene emerged as a particularly good candidate for further study.

Figure 3: GUN1 & GUN1^SG protein structure.

A, Schematic representation of GUN1, GUN1-mCherry and GUN1-mCherry-AKI. MTS, Mitochondrial Targeting Signal. AKI is GUN1 PTS1-Peroxisome Targeting Sequence. The I441N mutation present in GUN1^SG is indicated. B, GUN1 amino acid sequence. The MTS is highlighted in blue, the histidine of the catalytic site in green, the I441 in magenta and the AKI PTS1 in yellow. C, 3D structure of murine CAT (left) and 3D modelisation of GUN1 using I-TASSER modelling tool (right). The I441 is indicated by an arrow.

In order to explore the role of Pa_6_1340 in the ascospore germination process, a gene replacement was performed, in which the Pa_6_1340 CDS was substituted by a hygromycin B resistance marker (Figure S3). The gene disruption construct was introduced into a Δmus51::phleoR strain impaired for NHEJ (El-Khoury et al., 2008). Two independent hygromycin B-resistant [hygR] transformants were obtained. In order to purify ΔPa_6_1340::hygR from the Δmus51::phleoR mutation, we crossed both primary transformants with the S strain. Strikingly, we observed partially demelanized ascospores in the progeny of both crosses. Furthermore, when homokaryotic ascospores were sown on G+YE germination medium, only half of the progeny germinated, and those were only [hygS] melanized ascospores. This suggested that the [hygR] ΔPa_6_1340::hygR ascospores were the partially demelanized ones and that they were not able to germinate. These crosses were repeated on M2 medium supplemented with Tricyclazole, a fungicide impairing melanin synthesis and provoking spontaneous germination of ascospores in P. anserina (Coppin and Silar, 2007). We collected ascospores directly projected on M2 medium supplemented with hygromycin B and we isolated [hygR] germinating thalli. These thalli were fragmented and homokaryotic [hygR, phleoS] ΔPa_6_1340::hygR strains of each mating type were purified. Deletion of Pa_6_1340 CDS in these strains was verified by Southern Blot analysis (Figure S3) and mutant phenotypes were characterized. In homozygous ΔPa_6_1340::hygR X ΔPa_6_1340::hygR crosses ascospores exhibited demelanization and completely lost their ability to germinate on G+YE medium, a phenotype opposite to that of GUN1^SG (Figure 1). Genetic analyses of heterokaryotic ascospores showed that this phenotype due to ΔPa_6_1340::hygR deletion was recessive and that ΔPa_6_1340::hygR segregated with a Second Division Segregation (SDS) rate of 55% (see Mat. & Met.). Fertility in ΔPa_6_1340::hygR was affected too: perithecium production in a ΔPa_6_1340::hygR X ΔPa_6_1340::hygR cross was slightly reduced and ascospore production was significantly diminished compared to a wild-type cross (Figure S2). It has been shown that the formation of the appressorium in M. oryzae and the germination of melanized ascospores in P. anserina are two processes sharing common regulatory elements (Malagnac et al., 2008). We found that in the ΔPa_6_1340::hygR mutant, breaching of cellophane (a process involving appressorium development in P. anserina) was delayed compared to the wild-type (Table 2). However, microscopic observations did not detect any morphological defect of appressorium development (data not shown). Importantly, introduction of the wild-type allele of Pa_6_1340 carried on the pGUN1 plasmid (see Mat. & Met.) into the ΔPa_6_1340::hygR genome restored wild-type phenotypes thus showing that the deletion of Pa_6_1340 was responsible for the mutant phenotypes (Figure 1, 2, 4 & S2).

Figure 4: relative Carnitine-acetyltransferase (CAT) enzymatic activity in mycelial extracts.

CAT activity was assayed through the spectrophotometric measure of CoA-SH produced per minute per milligram of protein in cell extracts in presence of carnitine. Activities are reported as the activity ratio of the wild-type (WT) S strain. The CAT activity means and standard deviations have been calculated on 4 to 7 biological replicates (N is indicated for each genotype). Exact Two-Sample Fisher-Pitman Permutation Test has been realized to compare CAT activities. (*) CAT activities significantly different from the WT (P<0,05); (**) CAT activities in complemented strains significantly different from the activity in the respective mutant strains (P<0,05).

Finally, we addressed the question whether the phenotypes in the GUN1^SG mutants were due to the I441N mutation in Pa_6_1340 by testing whether ΔPa_6_1340::hygR and GUN1^SG are alleles of the same gene in a complementation test. In a first step, we genetically determined that spontaneous germination in GUN1^SG was a recessive trait, a prerequisite for the complementation test. These genetic analyses also showed that GUN1^SG segregated with a SDS rate of 54%, very similar to that of ΔPa_6_1340::hygR (55%), suggesting that ΔPa_6_1340::hygR and GUN1^SG could be allelic (see Mat. & Met.). We then crossed GUN1^SG with ΔPa_6_1340::hygR reasoning that if ΔPa_6_1340::hygR and GUN1^SG are allelic, no functional complementation in heterokaryotic ascospores in SDS asci is expected for the spontaneous germination of GUN1^SG: ΔPa_6_1340::hygR/GUN1^SG ascospores germinate spontaneously on M2 medium; in contrast, if GUN1^SG and ΔPa_6_1340::hygR are not allelic, functional complementation leading to restoration of wild-type phenotype is expected: Pa_6_1340::hygR/Pa_6_1340 GUN1^SG/GUN1 heterokaryotic ascospores in SDS asci germinate as wild-type. We observed that heterokaryotic ascospores in SDS asci germinated spontaneously on M2 medium showing that GUN1^SG and ΔPa_6_1340 were allelic (no complementation). This demonstrated that Pa_6_1340 was the gene mutated in the GUN1^SG mutant responsible for the spontaneous germination phenotype. We therefore named Pa_6_1340, GUN1. This evidence was confirmed when we showed that the GUN1^SG mutant was also complemented by ectopic integration of a wild-type copy of Pa_6_1340/GUN1 carried by the pGUN1 plasmid (Table 3 and Mat. & Met.).

Table 3: GUN1^SG X GUN1^SG pGUN1 (pBC-GUN1-genR).

42 homokaryotic ascospores were sown on both M2 and G+YE media. The numbers correspond to the amount of germinated spores.

P. anserina possess two CATs

A BLASTP search on the P. anserina predicted CDS database (http://podospora.i2bc.paris-saclay.fr/) identified a second CAT encoded by the Pa_3_7660 putative CDS. Compared to GUN1, this putative enzyme did not harbor any localization signal. Similarly to other fungi, P. anserina may be endowed with two types of CAT, one located in peroxisomes and in mitochondria (GUN1) and one remaining in the cytoplasm (Pa_3_7660). To confirm this hypothesis, we undertook a phylogenetical analysis of CATs in Eumycetes and searched the homologs of GUN1 in the genome sequence of representative fungal species (see Mat. & Met.). In these species, 2 CATs were always identified, except for S. cerevisiae in which three CATs were identified as previously shown (Swiegers et al., 2001). The protein sequences were aligned and the corresponding phylogenetic tree was built (Figure S4). The phylogeny of CATs in Eumycetes clearly indicated that there are 2 main types of CATs in fungi: the putative peroxisomal/mitochondrial CAT including GUN1, A. nidulans AcuJ and M. oryzae Pth2/Crat1 and the putative “cytoplasmic” CATs including P. anserina Pa_3_7760, A. nidulans FacC and M. oryzae Crat2. Careful analysis of protein sequences indicated that proteins of the former type all contained the appropriate sequence signals to locate in peroxisomes and in mitochondria. In addition, search for orthologs through OrthoDB (Kriventseva et al., 2019) did not identify orthologs for either GUN1 or Pa_3_7760 in plants nor in bacteria.

Pa_3_7760 putative CDS was renamed GUP1 for GUN1 Paralog 1. With the aim of determining the function of this second CAT and to assess its role in ascospore germination, we undertook a targeted gene disruption of GUP1. The Pa_3_7660 CDS was replaced by a phleomycin resistance marker through homologous recombination in a Δmus51::genR strain (see Mat. & Met. and Figure S5). The ΔGUP1 strain was purified from the Δmus51 mutation by crossing primary transformants with the wild-type S strain followed by the selection of [phleoR, genS] homokaryotic ascospores of the ΔGUP1 genotype in the progeny. ΔGUP1 ascospores had no melanization defect and germinated on G+YE medium as the wild-type. Importantly, when ΔGUP1 homokaryotic ascospores were sown on M2 medium, no spontaneous germination was observed, leading us to conclude that GUP1 deletion had no effect on ascospore melanization and germination in P. anserina. In addition, the ΔGUP1 strain differentiated wild-type mycelium and exhibited wild-type fertility, ascospore production, (Figure 2 & S2) appressorium development and cellophane breaching (data not shown). We then constructed the ΔGUN1 ΔGUP1 double mutant (see Mat. & Met.) and we observed that ΔGUN1 ΔGUP1 homokaryotic ascospores exhibited impaired melanization and lack of germination similarly to the ΔGUN1 ascospores.

GUN1 and GUP1 functions are conserved in fungi

Previous studies in fungi have revealed that CATs play an important role in primary metabolism and carbon source utilization. In particular, it has been shown that K.O strains of the cytoplasmic CAT do not grow on acetate whereas mutant strains of the peroxisomal/mitochondrial CAT do not grow on acetate and on media containing fatty acids such as oleic acid (Bhambra et al., 2006; Hynes et al., 2011; Liberti et al., 2013; Son et al., 2012; Swiegers et al., 2001). To assess the role of GUN1 and GUP1 in carbon source utilization, we have tested growth of the different mutant strains on acetate and oleic acid. As shown in Figure 2, the wild-type S strain was able to grow on all the tested media, including the Tween 40 control (Tween 40 is necessary to solubilize oleic acid), indicating that P. anserina was able to use this detergent as a carbon source. Remarkably, GUN1^SG as well as the GUN1^SG pGUN1 complemented strain grew as the wild-type. The ΔGUN1, ΔGUP1 and the double ΔGUN1 ΔGUP1 mutants grew as wild-type on dextrin (M2 medium), ΔGUN1 and ΔGUN1 ΔGUP1 exhibited slightly reduced growth on Tween 40, almost no growth on oleic acid and no growth on acetate whereas ΔGUP1 growth was impaired only on acetate. ΔGUP1 pGUP1 had restored wild-type growth on acetate indicating that wild-type GUP1 complemented ΔGUP1 deletion. This confirmed that GUP1 function was required in P. anserina on acetate. Similarly, wild-type growth was restored on Tween 40 and oleic acid in both ΔGUN1 pGUN1 and ΔGUN1 ΔGUP1 pGUN1 complemented strains indicating that GUN1 function was required for Tween 40 and oleic acid utilization. These data also showed that ΔGUN1 was epistatic on ΔGUP1 when P. anserina was grown on Tween 40 and on oleic acid. Strikingly, both the ΔGUN1 and the ΔGUN1 ΔGUP1 strains could grow on Tween 40 but not on oleic acid medium although this latter contained the same amount of Tween 40 (0,5%) (see Mat. & Met.). This suggested that impaired growth on oleic acid for ΔGUN1 and ΔGUN1 ΔGUP1 strains was due to a toxic effect of oleic acid in these mutant strains. Overall, these data showed that the roles of both main types of CATs were conserved in P. anserina: CATs of the AcuJ/Pth2/Crat1/GUN1-type are required for growth on acetate as well as on long chain fatty acids whereas CATs of the FacC/Crat2/GUP1-type are required for growth on acetate.

Structure prediction analysis of GUN1^SG loss-of-function

MAFFT alignment with the protein sequences encoded by the fungal orthologs of GUN1, including the ortholog from Homo sapiens revealed that the isoleucine 441 mutated in the GUN1^SG mutant (I441N) was highly conserved in Eumycota: it is conserved in Pezizomycotina, Saccharomycotina, Mucoromycota and in Basidiomycota (Figure S6). Using I-TASSER, we realized 3D structure prediction of GUN1, and we compared it to the 3D structure of the murine CAT (34% identity). As can be seen in Figure 3C, the overall structure of both enzymes was well conserved, showing that the 3D-modelling of GUN1 was congruent. Based on this GUN1 3D-model, we could localize the Isoleucine 441 near the extremity of an α-helix. Since hydrophobicity of amino acids is paramount in α-helix formation, we wondered whether the substitution of aliphatic isoleucine 441 by polar asparagine could destabilize the α-helix and/or the whole protein. We determined the Gibbs free-energy Gap (ΔΔG) provoked by the I441N substitution in GUN1^SG with STRUM (Funahashi et al., 2003). The calculated ΔΔG of 2.11 kcal.mol^-1 was indeed indicative of a destabilization of the GUN1^SG mutant protein but this low ΔΔG value (<6 kcal.mol^-1) was rather indicative of a local destabilization of the protein rather than a complete destabilization (Faure and Koonin, 2015). In line with this, we did not notice any stability issues in both chimera reporters GUN1^SG-mCherry and GUN1^SG-mCherry-AKI compared to GUN1-mCherry and GUN1-mCherry-AKI respectively, in our microscopic observations (see later). We also used the PROVEAN analysis tool to determine the impact of the I441N substitution on GUN1 function. Accordingly to the recessive nature of the GUN1^SG mutation, the calculated PROVEAN score of -6.63, far below the predefined cutoff of -2.5 was predictive of a “deleterious” loss-of-function effect of the I441N substitution on GUN1 function (Choi and Chan, 2015; Choi et al., 2012).

CAT activity decreases in GUN1^SG and ΔGUN1

We measured the CAT activity in different mutant strains in protein extracts from mycelium grown on M2 medium (Figure 4). It is worth to mention that we failed to measure CAT activity in ascospores and we could only obtain reliable results in mycelia (see Mat. & Met.). Compared to the wild-type, CAT activity in mycelium was greatly reduced in ΔGUN1, ΔGUN1 ΔGUP1 as well as the in GUN1^SG mutants. Significantly, CAT activity was restored to the wild-type level in the ΔGUN1 pGUN1 and ΔGUN1 ΔGUP1 pGUN1 complemented strains confirming that lack of GUN1 was responsible for reduced CAT activity. In the complemented GUN1^SG pGUN1 strain, the CAT activity was even higher than in the wild-type pointing to a role of CAT activity increase in the restoration of wild-type phenotype in GUN1^SG pGUN1. Interestingly, CAT activity in ΔGUP1 was similar to wild-type CAT activity, a result in agreement with previous observations in M. oryzae showing that CAT activity in ΔCrat2 (the ortholog of GUP1) mutants was not altered (Ramos-Pamplona and Naqvi, 2006). These results showing a decreased CAT activity in GUN1^SG and hence a loss-of–function of GUN1^SG were congruent with the modelized “deleterious” effect of the I441N mutation on GUN1^SG function and the recessivity of the GUN1^SG phenotype. However, the fact that similar reduced CAT activity was measured in GUN1^SG and ΔGUN1 (P<0.05) suggested that the CAT activity measured in mycelium did not account for the difference in phenotype between the GUN1^SG and ΔGUN1 mutants (i.e., germination and melanization of ascospores, acetate and acid oleic growth).

Subcellular localization of GUN1 and GUN1^SG proteins

Previous studies carried out on GUN1-type CATs in other fungal species have revealed that these enzymes could be localized in peroxisomes and in mitochondria (Hynes et al., 2011; Zhou and Lorenz, 2008). Analysis of GUN1 protein sequence using wolfPSORT indicated that GUN1 probably located in both peroxisomes and mitochondria. Accordingly, scanning GUN1 protein sequence with MitoFates allowed us to identify a Mitochondrial Targeting Sequence (MTS) and we manually identified the “AKI” tripeptide at the C-terminal end of the protein sequence as a type 1 Peroxisomal Targeting Sequence (PTS1) (Brocard and Hartig, 2006) (Figure 3). In order to investigate GUN1 subcellular localization as well as the impact of the GUN1^SG mutation on its subcellular localization, we tagged both GUN1 and GUN1^SG with mCherry and with mCherry-AKI, a modified mCherry version bearing the putative PTS1 peroxisome targeting signal of GUN1 in C-terminus (Figure 3). As previously mentioned, GUN1 is specifically induced in ascospores during germination (Demoor, unpublished data). To ensure that expression of the fusion proteins was under the control of the native GUN1 regulatory sequences, we tagged the endogenous GUN1 alleles (at the GUN1 locus) through insertion of the mCherry and mCherry-AKI coding sequences in 3’ of GUN1 and GUN1^SG CDS by homologous recombination (Figure S7 and Mat. & Met.). Four tagged strains were obtained: GUN1-mCherry, GUN1^SG-mCherry, GUN1-mCherry-AKI and GUN1^SG-mCherry-AKI. Importantly, GUN1-mCherry and GUN1-mCherry-AKI tagged strains germinated like the wild-type while GUN1^SG-mCHerry and GUN1^SG-mCHerry-AKI tagged strains germinated spontaneously on M2 medium like the GUN1^SG mutant. This suggested that the tagging by mCherry and mCherry-AKI did not modify GUN1 and GUN1^SG functions during ascospore germination. Each strain was crossed with strains expressing GFP markers tagging either mitochondria (mito-GFP) or peroxisomes (GFP-SKL) to obtain double tagged strains in the progeny (Ruprich-Robert et al., 2002; Sellem et al., 2007). Importantly, the presence of the mito-GFP or the GFP-SKL reporter genes did not modify ascospore germination. These double tagged strains allowed us to observe subcellular localization of GUN1 and GUN1^SG in mycelium (Figure 5) but not in melanized ascospores (Figure 6). For this purpose, each strain was crossed with the PaPks1¹³⁶ mutant producing partially demelanized ascospores in order to obtain all the double tagged strains in PaPks1¹³⁶ genetic background in the progeny. Contrary to the PaPks1¹⁹³ mutation which leads to spontaneous ascospore germination (Figure 1B), the PaPks1¹³⁶ mutation did not modify ascospore germination. All the PaPks1¹³⁶ double tagged strains produced partially demelanized ascospores allowing fluorescence microscopic observations within ascospores and were isolated in both mating types (mat+ and mat-) in order to proceed to homozygous crosses for ascospore production and observation. Ascospores were observed either in M2 liquid medium (non-induction condition) or in G liquid medium for induction of ascospore germination (Figure 6). We observed in ascospores and in mycelium that GUN1-mCherry-AKI and GUN^SG-mCherry-AKI could co-localize with both mito-GFP and GFP-SKL, showing that both GUN1- and GUN1^SG-mCherry-AKI reporter proteins could be found in mitochondria and in peroxisomes (Figure 5). Careful comparison of GFP-tagging pattern and mCherry tagging pattern indicated that GUN1-mCherry-AKI and GUN^SG-mCherry-AKI could be absent in some mitochondria or in some peroxisomes. This was especially striking in “non-induction” condition (liquid M2) in GUN1-mCherry-AKI mito-GFP ascospores where no GUN1-mCherry-AKI co-localized with mitochondria (Figure 6). This data suggested that the distribution of GUN1-mCherry-AKI between mitochondria and peroxisomes might change upon germination induction, GUN1-mCherry-AKI localizing more frequently in mitochondria when ascospores germinate. In line with this, GUN1^SG-mCherry-AKI co-localization with mito-GFP was always important in mycelium and in ascospores, these latter germinating spontaneously in both M2 and G liquid medium.

Figure 5: Spinning disk imaging of GUN1 and GUN^SG subcellular localization in mycelium.

GUN1 and GUN1^SG have been tagged with the mCherry or the mCherry-AKI (bearing GUN1 PTS1 AKI triad) fused in C-terminus. A) Every strain analyzed carry the GFP-SKL marker tagging the peroxisomes. B) Every strain carry the mito-GFP reporter tagging mitochondria. Scale bar 5 μm.

Figure 6: Spinning disc imaging of GUN1-mCherry-AKI and GUN1^SG-mCherry-AKI in ascospores.

All the ascospores imaged carry the PaPks1¹³⁶ mutation partially impairing ascospore melanisation and making ascospores transparent for fluorescence imaging. A) Every strain carry the GFP-SKL reporter tagging peroxisomes. B) Every strain carry the mito-GFP reporter tagging mitochondria. M2 liquid medium: no induction of ascospore germination. G liquid medium: ascospore germination induction. Scale bar 5 μm

We observed that GUN1-mCherry and GUN1^SG-mCherry co-localized only with the mito-GFP reporter in ascospores (Figure S8) as well as in mycelium (data not shown). This observation was consistent with previous studies on the localization of G. zeae CAT1-GFP (the ortholog of GUN1) showing that adding the GFP in C-terminus of the protein masked the PTS1 signal of CAT1 (Son et al., 2012). More generally, it has been shown that adding a tag after a C-terminal PTS1 signal abolishes import into peroxisomes, suggesting that GUN1-mCherry and GUN1^SG-mCherry could be mislocalized (Ast et al., 2013; Hooks et al., 2012). It is worth noting that ascospores in GUN1-mCherry tagged strain germinated as wild-type and ascospores in GUN1^SG-mCherry tagged strain germinated spontaneously strongly suggesting that mislocalization of GUN1- or of GUN1^SG-mCherry did not affect ascospore germination. In contrast, whereas GUN1-mCherry-AKI and GUN^SG-mCherry-AKI strains grew as the wild-type on the different media tested, the GUN1-mCherry and GUN^SG-mCherry strains did not grow on oleic acid (Table S3). This result suggested that peroxisomal localization of GUN1 (and GUN1^SG) was required for oleic acid metabolism.

Table S2: primers list

Table S3: Growth of mCherry and mCherry-AKI tagged strains on different carbon sources.

All the media have the same composition, except for the carbon source. The Tween 40 control medium and the oleic acid medium contain 0,5% Tween 40. +, wild-type growth; -, altered growth

GUN1 acts upstream of the MAPK PaMpk2 pathway and of the PaNox2/PaPls1 complex

We have shown in previous studies that the MAPK PaMpk2 pathway, PaNox2, its regulatory subunit PaNoxR and PaPls1 are essential for ascospore germination in P. anserina : the deletion of these genes blocks ascospore germination (Brun et al., 2009; Lalucque et al., 2012; Lambou et al., 2008; Malagnac et al., 2004). We took advantage of the spontaneous germination phenotype of the GUN1^SG mutant to conduct epistasis studies in order to place GUN1 in the regulatory cascade triggering ascospore germination. These studies were carried out by crossing GUN1^SG with the ΔPaMpk2, ΔPaPls1 and ΔPaNox2 strains. 48 homokaryotic spores were sown on M2 medium for each cross, but none of the germinated spores presented the ΔPaMpk2, ΔPaPls1 or ΔPaNox2 deletions (Table 4). This clearly showed that PaMpk2, PaPls1 and PaNox2 were required for germination in GUN1^SG ascospores and suggested that GUN1 acts upstream of PaMpk2, PaNox2 and PaPls1 in the cascade controlling ascospore germination. To confirm that GUN1 was upstream of the PaMpk2 MAPK pathway, we crossed the ΔGUN1 strain with the PaMKK2^c mutant carrying a constitutively active allele of PaMKK2. It has been previously shown that the PaMKK2^c mutation induces phosphorylation of PaMpk2 and spontaneous ascospore germination (Lalucque et al., 2012) (Figure 7). In the progeny of this cross, 40 homokaryotic ascospores were sown on M2 medium (Table 5). Every germinated ascospore carried PaMKK2^c, thus indicating that spontaneous germination on M2 medium was due to PaMKK2^c. Strikingly, ΔGUN1 MKK2^c ascospores (26/40) germinated on M2 medium, demonstrating that MKK2^c-induced ascospore germination was not blocked by the inactivation of GUN1. This confirmed that GUN1 was upstream of the MAPK PaMpk2 pathway in ascospore germination regulation. In line with this, we found that PaMpk2 was phosphorylated in GUN1^SG ascospores at a comparable level as in PaMkk2^c ascospores and as in wild-type ascospores induced for germination (Figure 7). Altogether, these data clearly indicated that GUN1^SG triggered spontaneous ascospore germination through the activation of the PaMpk2 MAPK pathway (Figure 8).

Table 4: Results of the crosses between GUN1^SG and ΔPaPls1, ΔPaNox2, ΔPaMpk2.

48 homokaryotic ascospores were sown on G+YE medium for each cross. The numbers correspond to the amount of germinated spores.

Figure 7. Western blot analysis of PaMpk2 phosphorylation in ascospores.

Proteins were extracted from genetically homogenous ascospores. Anti-p44 and anti-phospho-p44 antibodies recognizing both PaMpk1, PaMpk2 and p-PaMpk1 and p-PaMpk2 respectively were used. For ascospore germination (WT ind.) induction, ascospores where placed on G+YE medium for 2h before protein extraction (see Mat. & Met.). PaMKK2^c constitutive mutation induces PaMpk2 phosphorylation and spontaneous ascospore germination.

Table 5: PaMKK2^C::phleoR X ΔGUN1::hygR.

40 homokaryotic ascospores were sown on M2 medium and genotyped.

Figure 8. Regulation of ascospore germination.

Breaking of dormancy is initiated by germination triggers: ammonium acetate and bactopeptone in vitro. The activities of the PTS1-type and PTS-2 type importomer components Pex5, Pex7 and Pex13 are required for germination. We speculate that GUN1-driven acetyl-CoA shuttling to mitochondria activates the Mpk2/Fus3 MAPK pathway which in turn activates the NADPH oxidase complex Nox2-Pls1-NoxR. Whether activation of the Nox2 complex sets up a septin ring and actin cytoskeleton rearrangement at the germination pore in a similar manner as in M. oryzae appressorium remains to be addressed.

GUN1^SG induces spontaneous ascospore germination independently of Pex7

Peroxisomes are key organelles for germination. It has been shown that mutants of peroxisomal importomers such as ΔPex5, ΔPex7 and ΔPex13 produce partially demelanized ascospores impaired for germination (Peraza Reyes and Berteaux-Lecellier, 2013; Peraza-Reyes et al., 2011). We crossed GUN1^SG with the ΔPex5 ΔPex7 double mutant and with the ΔPex13 mutant and we checked whether ΔPex5 GUN1^SG, ΔPex7 GUN1^SG and ΔPex13 GUN1^SG double mutants could germinate when sown on G+YE germination medium (Table 6 & 7). None of the germinated ascospores carried ΔPex5 or ΔPex13, suggesting that ΔPex5, ΔPex13, ΔPex5 GUN1^SG and ΔPex13 GUN1^SG ascospores did not germinate. The fact that ΔPex5 and ΔPex13 were epistatic over GUN1^SG indicated that the function of both genes was required for GUN1^SG-induced ascospore germination. Very interestingly, 15 ascospores carrying ΔPex7 germinated on G+YE germination medium. To test whether they also carried the GUN1^SG mutation, we analyzed spontaneous germination in the progeny of the mating tests performed to genotype spores (crossing with wild-type S). Abundant spontaneous germination was observed in bulk in this progeny showing that the 15 ascospores isolated were of the ΔPex7 GUN1^SG genotype and not ΔPex7. In other words, GUN1^SG suppressed the ΔPex7 ascospore germination defect. This result indicated that germination was abolished in ΔPex7 ascospores (as previously shown) and that GUN1^SG was epistatic over ΔPex7, inducing germination independently of Pex7.

Table 6: ΔPex5 ΔPex7 (ΔPex5::hygR ΔPex7::phleoR) X GUN1^SG.

75 homokaryotic ascospores were sown on G+YE medium. *, ascospores of this genotype show spontaneous germination on standard M2 medium.

Table 7: ΔPex13 (ΔPex13::hygR) X GUN1^SG.

64 homokaryotic ascospores were sown on G+YE medium. *, ascospores of this genotype show spontaneous germination on standard M2 medium.

Discussion

Despite its importance in the fungal life cycle, the regulation of ascospore germination in filamentous fungi has been poorly investigated so far. In this study, we aimed at uncovering new actors of this regulation pathway in P. anserina. To that end, we conducted a direct genetic screen, a particularly powerful approach to identify new genes. A majority of the mutants isolated, including all suppressors of ΔPaNox2 and ΔPaPls1, showed spontaneous but abnormal ascospore germination. However, for six mutants, germination proceeded as in the wild-type through the germination pore at the tip of the ascospore. We hypothesized that in these mutants, ascospore dormancy was broken and that these were mutants specifically impaired in the control of ascospore germination. In this paper, we characterize the first of these six Germination UNcontrolled-GUN mutants, GUN1^SG. Although most of the spontaneous germination analyses were performed on M2 medium containing dextrin, the GUN1^SG mutant germinated on medium lacking carbon and nitrate sources indicating that in this mutant, control of dormancy escaped possible nutrient stimuli (data not shown). Through whole genome sequencing and genetic analyses, we showed that the gene mutated in GUN1^SG is the Pa_6_1340 putative CDS. This CDS encodes a peroxisomal/mitochondrial Carnitine-acetyltransferase (CAT), a key metabolic enzyme involved in acetyl-CoA shuttling between peroxisomes and mitochondria (Elgersma et al., 1995; Strijbis and Distel, 2010). Gene expression of this CAT is significantly induced during ascospore germination, highlighting the pivotal role of this enzyme during this process (Demoor, unpublished data). The mutation in GUN1^SG CDS is a substitution of the conserved isoleucine 441 by an asparagin (I441N). 3-D modelisation of GUN1 and of mutated GUN1^SG proteins combined to in silico analyses of the stability of GUN1^SG predict a moderate effect of the I441N substitution on the overall stability of the protein but a deleterious effect on its activity. This prediction correlates with the recessive nature of the GUN1^SG mutation as well as the reduced CAT activity measured in GUN1^SG, both pointing to a loss-of-function of the GUN1^SG allele. Hence, GUN1 may act as an ascospore germination inhibitor. However, we also show that deletion of GUN1 leads to complete lack of germination and defect in melanin synthesis, indicating that GUN1 function is required for both processes and suggesting that the GUN1^SG allele is hypomorphic compared to ΔGUN1 which is a null allele. Impairment of both germination and melanization are frequently observed in mutants of peroxisomal import machinery as well as in mutants of peroxisomal/mitochondrial β–oxidation (Boisnard et al., 2009; Peraza Reyes and Berteaux-Lecellier, 2013; Ruprich-Robert et al., 2002). Indeed, ascospores in the ΔEchA mutant impaired for mitochondrial β-oxidation and in the ΔFox2 mutant impaired for peroxisomal β–oxidation, show reduced rate of germination. Interestingly, ΔGUN1 exhibits complete lack of germination, suggesting that GUN1 and acetyl-CoA shuttling between peroxisomes and mitochondria is essential during activation of ascospore germination. As in many other fungi, we found a second CAT encoded in P. anserina genome. Deletion of this second CAT encoding gene, Pa_3_7660/GUP1 (GUN1 Paralog 1), does not show any ascospore germination defect nor melanization defect, suggesting that this CAT has no role in both processes. Furthermore, while CAT activity in ΔGUN1 and in GUN1^SG are reduced in mycelium, CAT activity in ΔGUP1 resembles that of the wild-type, a situation similar to M. oryzae. In this plant pathogen, while the GUN1 ortholog Pth2 mutants show reduced CAT activity and reduced pathogenicity, the ΔCrat2 K.O strain (GUP1 ortholog) exhibits wild-type CAT activity and pathogenicity (Ramos-Pamplona and Naqvi, 2006).

As previously found in fungi (M. oryzae, A. nidulans, G. zeae and S. cerevisiae), we show that in P. anserina, the peroxisomal/mitochondrial CAT GUN1 is required for growth on acetate and oleic acid while the cytosolic CAT GUP1 is required for growth on acetate only, showing strong conservation of the function of both enzymes in fungal primary metabolism (Bhambra et al., 2006; Hynes et al., 2011; Liberti et al., 2013; Son et al., 2012; Swiegers et al., 2001). Nonetheless, GUN1 is required for growth on fatty acids, but we also show that oleic acid is toxic for ΔGUN1 mutants, as previously demonstrated for Pex2^- mutants impaired for peroxisomal import (Ruprich-Robert et al., 2002). Indeed, both ΔGUN1 and ΔGUN1 ΔGUP1 strains grow better on Tween 40 control medium than on oleic acid medium containing similar amount of Tween 40. In contrast to peroxisomal import mutants such as Pex2^-, which are sterile in homozygous cross, fertility and ascospore production are only moderately decreased in ΔGUN1 and ΔGUN1 ΔGUP1.

With the aim to understand how the mutation in GUN1^SG triggers breaking of dormancy, we explored both GUN1^SG CAT activity and GUN1^SG subcellular localization. GUN1^SG shows a similar loss of CAT activity in mycelium to that of ΔGUN1. However, ΔGUN1 and GUN1^SG mutant strains exhibit noticeable phenotype discrepancies: ΔGUN1 produces non-germinating demelanized ascospores while GUN1^SG produces spontaneously-germinating melanized ascospores and GUN1^SG grows as the wild-type on both oleic acid and acetate but not ΔGUN1. We were not able to measure CAT activity in ascospores and therefore to properly address the question of CAT activity during germination in GUN1^SG and ΔGUN1.

To explore how GUN1^SG causes spontaneous germination, both the wild-type GUN1 protein and the mutant GUN1^SG protein were tagged with mCherry or mCherry-AKI (AKI is the PTS1 peroxisomal import signal present in C-terminus of GUN1) and co-localization studies with GFP-tagged peroxisomes and GFP-tagged mitochondria were performed. Our data show that in mycelium as well as in ascospores, GUN1 is located both in peroxisomes and in mitochondria. This dual localization is in agreement i) with the predicted localization of this CAT bearing both a Mitochondrial Targeting Sequence (MTS) in N-terminus and the Peroxisomal Targeting Sequence (PTS1) AKI in C-terminus and ii) with studies of GUN1 orthologs in other fungi such as A. nidulans and G. zeae (Hynes et al., 2011; Son et al., 2012). In contrast, M. oryzae Pth2 was shown to only localize in peroxisomes (Bhambra et al., 2006). Attention must be drawn to the fact that there are no differences between GUN1 and GUN1^SG subcellular localization observed in mycelium as well as in ascospores subjected to germination induction: both proteins being more or less equally distributed between mitochondria and peroxisomes. Nevertheless, GUN1 seems to be almost exclusively located in peroxisomes in dormant ascospores not subjected to germination trigger. These data suggest that the breaking of dormancy in wild-type and in GUN1^SG ascospores may involve shuttling of GUN1^SG from peroxisomes to mitochondria. We show that, in keeping with the role of mitochondria in ascospore germination, the mislocalization of GUN1-mCherry (and GUN1^SG-mCherry) solely in mitochondria does not impair germination neither melanization of ascospores. However, GUN1-mCherry and GUN1^SG-mCherry strains do not grow on oleic acid, in the same way as ΔGUN1. A comparable effect has been reported in A. nidulans where mislocalized AcuJ in cytoplasm impairs growth on oleic acid but not on acetate (Hynes et al., 2011). These data point to a central role of peroxisomal localization of GUN1 in oleic acid utilization independent of ascospore germination. However, it has been shown that peroxisomes are essential for germination in P. anserina and our data confirm that ΔPex5 and ΔPex13 strains lacking PTS1-dependent peroxisomal import machinery as well as Δpex7 strains lacking PTS2-dependent peroxisomal import machinery cannot germinate (Peraza Reyes and Berteaux-Lecellier, 2013; Peraza-Reyes et al., 2011). How ascospores carrying mislocalized GUN1 germinate is an open question. Remarkably, we show that GUN1^SG can induce spontaneous ascospore germination independently of Pex7 and the PTS2-dependent peroxisomal import. Given that GUN1 bears a PTS1-AKI in C-terminus (and no internal PTS2) and that ΔPex5 is epistatic over GUN1^SG, it is highly likely that GUN1 (and GUN1^SG) peroxisomal import is dependent on the PTS1 and not on the PTS2 import pathway.

Through the characterization of the GUN mutants, we aimed at uncovering new genes that control dormancy in ascospores. Genetic approaches in P. anserina and in particular the amenability to performing epistasis studies makes this model fungus a powerful system to decipher regulation pathways such as the one controlling ascospore germination. In this paper, we investigated the relationships between GUN1 and already known actors of ascospore germination: the PaMpk2 MAPK pathway, the tetraspanin Pls1 and the NADPH oxidase Nox2 complex (Lalucque et al., 2012; Lambou et al., 2008; Malagnac et al., 2004). We show that GUN1^SG requires PaMpk2, PaNox2 and PaPls1 functions to induce spontaneous germination of ascospores. Conversely, the MAPKK constitutive PaMKK2^c allele inducing PaMpk2 phosphorylation and spontaneous ascospore germination does not require GUN1 function. Furthermore, we show that GUN1^SG controls the PaMpk2 pathway by activating PaMpk2 phosphorylation. Altogether, these data demonstrate that GUN1 acts upstream of the PaMpk2 pathway and the PaNox2/PaPls1 complex in the regulatory cascade controlling ascospore germination (Figure 8).

Melanin is a major component of appressorium cell wall in M. oryzae where it is involved in turgor pressure maintenance (Gómez and Nosanchuk, 2003). M. oryzae mutants such as Pth2^- showing impairment of melanin synthesis cannot build up the turgor pressure necessary in the appressorium for host-penetration and are therefore non-pathogenic. As observed in the PaPks1¹⁹³ mutant devoid of melanin biosynthesis, melanin in ascospores is important to avoid uncontrolled “accidental” germination (Coppin and Silar, 2007). The melanization defect exhibited by the ΔGUN1 ascospores is likely due to lack of acetate supply to the dihydroxynaphthalene pathway involved in melanin biosynthesis, a defect shared by the ΔechA and Δfox2 P. anserina mutants, impaired in mitochondrial and in peroxisomal β-oxidation respectively (Boisnard et al., 2009; Coppin and Silar, 2007). But unlike ΔechA and Δfox2 ascospores that germinate spontaneously, ΔGUN1 ascospores do not, suggesting that the melanization defect in ΔGUN1 ascospores is not sufficient to induce spontaneous germination. Accordingly, Tricyclazole, a fungicide inhibiting melanin biosynthesis or the PaPks1¹⁹³ mutation blocking melanin production (Coppin and Silar, 2007), suppress the germination defect of ΔGUN1 ascospores. Indeed, PaPks1¹⁹³ ΔGUN1 ascospores germinate spontaneously (data not shown). Hence, it is likely that in ΔGUN1 ascospores, residual melanin in cell wall is enough to avoid “accidental” spontaneous germination.

The fact that Tricyclazole and PaPks¹⁹³ null mutation trigger germination of ΔGUN1 ascospores suggests that these ascospores are competent for formation of the germination peg and eventually hyphal growth but that they might be blocked in the formation of the germination pore. The formation of a pore in melanized ascopores is a process sharing similarities with the formation of the pore in M. oryzae appressorium (Malagnac et al., 2008). In this pathogenic fungus, the formation of the appressorial pore is preceded by the formation of a septin ring required for actin cytoskeleton remodeling and appressorial pore solidity. In-depth studies in M. oryzae have deciphered the intricate genetic signaling, culminating in the setting up of this septin ring (Dagdas et al., 2012; Ryder et al., 2019). Among other regulatory components the Fus3/Pmk1/PaMpk2 pathway and the NoxB/Pls1 complex play a key role in septin ring assembly. The fact that both pathways are also essential for ascospore germination in P. anserina, N. crassa and S. macrospora, three species producing melanized ascospores lead us to hypothesize that a similar process involving septin ring assembly and cytoskeleton remodeling may take place to initiate the formation of the germination pore. Given the similarities in the regulation of appressorium functioning and ascospore germination when those are melanized, we speculate that studying and discovering new genes controlling ascospore germination in P. anserina may lead to the discovery of new pathogenesis factors controlling appressorium development in pathogenic fungi. The characterization of GUN1, the ortholog of M. oryzae Pth2 represents a proof of concept. The characterization of the other GUN mutants and in the future, the isolation of new GUN mutants will be of great interest to better understand ascospore germination but also to discover new pathogenic factors, killing two birds with the same stone.

Materials and Methods

Strains and culture conditions

The strains used in this study are all listed in Table 1. All of these P. anserina strains derive from the wild-type S strain, ensuring a homogenous genetic background (Espagne et al., 2008; Rizet, 1952). The PaPks1¹⁹³ mutant for the polyketide synthase encoding gene acting at the first step of melanin synthesis is described in (Coppin and Silar, 2007). Standard culture conditions, media and genetic methods for P. anserina were described in (Rizet and Engelmann, 1949) and can be found in (Silar, 2020) and on the Podospora data base http://podospora.i2bc.paris-saclay.fr/. The composition of the M0 and M3 media is similar to that of the M2 medium, except that dextrin is replaced by glucose in the M3 medium (5,5 g.L^-1), while no carbon source is added in the M0 medium. This M0 medium was used as a basis for the development of media in which the only carbon source was sodium acetate (60 mM) or oleic acid (Sigma-Aldrich) (6 mM) dissolved in Tween 40 (0.5 %). A control medium M0 with only Tween 40 (0.5%) was also used. The germination medium used in this study was supplemented with Yeast extract (G+YE) 5 g.L^-1. In order to allow germination in strains producing ascospores unable to germinate, crosses were set up on M2 medium supplemented with Tricyclazole (1 μg.mL^-1), a fungicide impairing melanin synthesis in P. anserina ascospores (Coppin and Silar, 2007).

Genetic screening of constitutively germinating mutants

It has been shown that wild-type P. anserina ascospores do not germinate on standard M2 medium and that the ΔPaNox2 and ΔPls1 mutant strains produce ascospores unable to germinate on all tested media (Lambou et al., 2008; Malagnac et al., 2004). With the aim to isolate mutants producing spontaneously germinating ascospores, a UV mutagenesis was performed on auto-fertile mat-/mat+, wild-type S, ΔPaNox2 and ΔPls1 strains. The selection process of the germination mutants is summarized in Figure S1. Shortly after UV exposure, followed by a one-day culture in the dark to prevent repair by photoreactivation, the mutated strains were grown on standard M2 medium for one week until they developed mature ascospores-producing perithecia. In order to recover spontaneously germinating ascospores, M2 medium plates were put on top of the plates bearing perithecia, which allowed ascospores to be harvested in bulk on M2 medium. In P. anserina, most of the progeny is composed of heterokaryotic mat+/mat- ascospores leading to self-fertile mycelium upon germination (Silar, 2013). The thalli produced by the spontaneously-germinating ascospores were incubated until they formed mature perithecia projecting their ascospores. Ascospores produced by these perithecia were individually collected with a needle and transplanted onto M2 medium. To ensure their independence, a single mutant (i.e., a single spontaneously-germinating homokaryotic ascospore) per initial plate was selected for further analyses. For every mutant, progeny analysis of mutant X wild-type S crosses showed that a single mutated locus was responsible for the mutant phenotype (i.e., spontaneous germination of ascospores). For mutants recovered with the ΔPaNox2 and ΔPls1 strains, genetic analyses showed that the mutations enabling germination were unlinked to the ΔPaNox2 and ΔPls1 mutations, respectively. Homokaryotic mat- and mat+ mutant strains were isolated from the progenies and homozygous mutant crosses were performed to check for the fertility/sterility of the mutants. For every isolated strain, microscopic observations were also performed to determine whether the ascospores germinated through the germination pore at the tip of the ascospore or through any other part of the ascospore (data not shown).

Tetrad analysis in the GUN1^SGand in the ΔGUN1 strains

Demonstration of the recessivity of the GUN1^SG allele

P. anserina produces mainly asci containing four heterokaryotic/dikaryotic ascospores, allowing non-ordered tetrad analysis of first division segregation (FDS) asci and second division segregation (SDS) asci. To determine dominance/recessivity of the GUN1^SG allele, we crossed the GUN1^SG mutant with the WT. In thirty asci of the progeny, we found that 16 asci contained four heterokaryotic ascospores unable to germinate on M2 medium (4 [non-germinating] ascospores) and 14 asci contained different number of ascospores germinating spontaneously: 3 [non-germinating]; 1 [germinating] or 2 [non-germinating]; 2 [germinating] ascospores. Importantly, we never observed asci containing more than 2 ascospores germinating on M2 medium. Assuming some ascospore germination failure due to their manipulation, especially the ones germinating spontaneously, we concluded that i) asci of the first type were SDS asci containing 4 GUN1^SG/GUN1 ascospores of the [WT] phenotype (non-germinating on M2 medium), ii) asci of the second type were FDS asci containing 2 GUN1^SG /GUN1^SG of the [GUN] Germination UNcontrolled phenotype (germinating on M2 medium) and 2 GUN1/GUN1 ascospores of the [WT] phenotype. The GUN1^SG allele was thus recessive against the wild-type GUN1 allele (i.e., only GUN1^SG/GUN1^SG ascospores germinated on M2 medium but not the GUN1^SG/GUN1 ones).

Demonstration of the recessivity of the ΔPa_6_1340::hygR (ΔGUN1) allele

The recessivity of the ΔPa_6_1340::hygR (ΔGUN1) phenotypes was tested by crossing ΔPa_6_1340::hygR with the wild-type. We sowed 20 asci on G+YE germination medium and we obtained i) 9 asci containing 2 [demelanized, non-germinating] and 2 [melanized, germinating, hygS] ascospores; these were identified as First Division Segregation (FDS) asci containing 2 ΔPa_6_1340::hygR/ΔPa_6_1340::hygR and 2 wild-type Pa_6_1340/Pa_6_1340 ascospores respectively. The 11 other asci contained 4 [melanized, germinating, hygR] ascospores which were identified as SDS asci composed of 4 ΔPa_6_1340::hygR/Pa_6_1340 ascospores. These data demonstrated that the phenotypes due to ΔPa_6_1340::hygR deletion were recessive and that SDS rate for the Pa_6_1340 locus was 55 %.

Complementation test between the ΔPa_6_1340::hygR (ΔGUN1) and the GUN1^SG alleles

We crossed GUN1^SG with ΔPa_6_1340::hygR and we reasoned that if ΔPa_6_1340::hygR and GUN1^SG are allelic, no functional complementation in heterokaryotic ascospores in SDS asci is expected for the spontaneous germination of GUN1^SG: ΔPa_6_1340::hygR/GUN1^SG ascospores germinate spontaneously on M2 medium; in contrast, if GUN1^SG and ΔPa_6_1340::hygR are not allelic, functional complementation leading to restoration of wild-type phenotype is expected: Pa_6_1340::hygR/Pa_6_1340 GUN1^SG/GUN1 heterokaryotic ascospores in SDS asci germinate only on G+YE medium but not on M2 medium. The SDS asci (54%) (n=50) were easily recognized since they contained 4 [hygR, melanized] ascospores, compared to the FDS asci, containing 2 [hygS, melanized] ascospores and 2 [non germinating, demelanized] ascospores. Strikingly, we observed that heterokaryotic ascospores in SDS asci germinated spontaneously on M2 medium showing that GUN1^SG and ΔPa_6_1340 were allelic. This demonstrated that Pa_6_1340 was the gene mutated in the GUN1^SG mutant responsible for the spontaneous germination phenotype.

GUN1^SG genome sequencing and analysis

In a first step towards identifying the gene mutated in GUN1^SG through whole genome sequencing, we backcrossed the mutant for five generations with the parental wild-type S strain as to eliminate any mutation unrelated to the mutant phenotype. The GUN1^SG genomic DNA was then extracted as described in (Lecellier and Silar, 1994). The genomic DNA was then subjected to complete sequencing with the Illumina technology at the Imagif facility, Gif-sur-Yvette (CNRS, I2BC Sequencing Facility, https://www.i2bc.paris-saclay.fr/spip.php?article1184&lang=en). Custom-made libraries had 300 bp inserts and sequencing was 76-bp paired-end. Coverage was 80-fold. The sequence reads were then mapped onto the latest version of the reference genome of the S strain (Grognet et al., 2014). Potential mutations were detected using Samtools and bcftools on the Galaxy web server (https://usegalaxy.org/).

Deletion of GUN1, GUP1 and construction of the double ΔGUN1 ΔGUP1 strain

ΔGUN1

the deletion of Pa_6_1340/GUN1 and its paralog Pa_3_7660/GUP1 were performed using deletion cassettes made of two overlapping PCR fragments (Figure S3 & S5) (Lalucque et al., 2012). This method is based on the generation of two DNA PCR fragments carrying a resistance marker flanked by either 5′ or 3′ flanking sequences of the targeted gene. For Pa_6_1340/GUN1, we first amplified the 803 bp-long 5′ and 486 bp-long 3′ flanking regions of the S strain DNA by PCR with primers pairs: 1340_1/1340_2 and 1340_3/1340_4 respectively (Table S2). At the same time, the hygromycin B resistance marker was amplified with 1340_MkF and 1340_MkR (Table S2) from the pBC-hygR vector (Silar, 1995). In a second PCR round, using primers 1340_1 and 1340-MkR, and 1340-MkF and 1340_4, the resistance marker was fused with the 5′ and with the 3′ flanking regions respectively. Both PCR products were used to transform a Δmus51::genR strain, in which the mus51 gene involved in the NHEJ repair system is replaced by a geneticin resistance gene [genR], allowing high rate of homologous recombination (El-Khoury et al., 2008). Two hygromycin B-resistant [hygR] transformants were obtained. Each one was crossed with the wild-type S strain. We observed in the progeny that homokaryotic [hygR] ascospores did not germinate. Consequently, crosses were performed on M2 medium supplemented with Tricyclazole, leading to spontaneous germination of ascospores (Coppin and Silar, 2007). The [hygR] thalli coming from spontaneously germinating ascospores were selected on M2 medium supplemented with hygromycin B, fragmented and [hygR, phleoS] ΔGUN1::hygR homokaryotic mycelia of each mating type were isolated. Deletion of Pa_6_1340 was verified by Southern blot analysis (Figure S3). Only one strain was selected for further analyses.

ΔGUP1

the same protocol was performed to produce the deletion cassettes for Pa_3_7660/GUP1. Using the primers pairs: 7660_F1/7660_R2 and 7660_R3/7660_R4 (Table S2), the 1104 bp-long 5′ and 1011 bp-long 3′ Pa_3_7660 flanking regions were PCR-amplified, while the phleomycin resistance marker was amplified with primers 7660_MkF and 7660_MkR (Table S2) from a pBC-phleoR plasmid (Silar, 1995). In a second PCR round, using primers 7660_F1 and 7660_MkR, and 7660_MkF and 7660_R4 the resistance marker was fused with the 5′ and the 3′ flanking regions. Both PCR products were used to transform a Δmus51::genR strain. 26 phleomycin-resistant [phleoR] transformants were obtained and two independent [phleoR, genS] ΔGUP1::phleoR strains were selected from the progeny of a cross with the wild-type S strain (germination of ΔGUP1::phleoR ascospores was as wild-type). Deletion in these two independent strains was verified by Southern blot analysis (Figure S5). Only one strain was selected for further analyses.

ΔGUN1 ΔGUP1

with the aim to construct the ΔGUN1 ΔGUP1 double mutant, we crossed ΔGUN1 with ΔGUP1 on M2 supplemented with Tricyclazole, we selected [hygR, phleoR] mycelia from spontaneously germinating ascospores on M2 medium supplemented with hygromycin and phleomycin. These mycelia were fragmented and homokaryotic [hygR, phleoR] ΔGUN1 ΔGUP1 strain of each mating type was isolated.

Plasmid constructions for the complementation of GUN1^SG, ΔGUN1 and ΔGUP1

Construction of pGUN1

The Pa_6_1340/GUN1 CDS, its 803 bp 5’ upstream and 486 bp 3’ downstream sequences were amplified by PCR from wild-type S genomic DNA using 1340_1 and 1340_4 primers (Table S2). The PCR product obtained was cloned blunt-end into pBC-genR plasmid carrying a geneticin resistance marker digested by EcoRV to produce the pBC-GUN1-genR plasmid (renamed pGUN1 for sake of simplicity). The insert was verified by sequencing (data not shown). This plasmid was used to transform the ΔGUN1::hygR deletion strain. 2 [genR] transformants were obtained and checked for the restoration of wild-type phenotypes in ascospores. To that end, two of these transformants were crossed with the ΔGUN1::hygR strain. In the progeny of both crosses, [hygR, genR] melanized homokaryotic ascospores germinated (on G+YE germination medium) allowing us to purify ΔGUN1 pGUN1 mat+ and mat-homokaryotic strains and to show that wild-type GUN1 complemented the ΔGUN1 mutation. [hygS, genR] GUN1 pGUN1 homokaryotic ascospores were also isolated in the progeny. These ascospores germinated as the wild-type. The pGUN1 was also used to transform the GUN1^SG mutant. 3 [genR] transformants were obtained and crossed with GUN1^SG to assess restoration of wild-type germination in the progeny. For one transformant, we observed that GUN1^SG pGUN1 progeny showed wild-type ascospore germination, i.e GUN1^SG pGUN1 spores did not germinate spontaneously on M2 medium but germinated on G+YE germination medium, showing that ectopic wild-type GUN1 complemented the GUN1^SG mutant (Table 3).

Construction of pGUP1

the Pa_3_7660/GUP1 CDS, its 1104 bp 5’upstream and 1011 bp 3’ dowstream sequences were amplified by PCR from wild-type S genomic DNA using primers 7660_F1 and 7660_R4 (Table S2). The PCR product obtained was cloned blunt-end into pBC-nouR (carrying a nourseothricin resistance marker) digested by EcoRV to produce the pBC-GUP1-nouR plasmid (renamed pGUP1). The pGUP1 plasmid was used to transform the ΔGUP1::phleoR deletion strain. 17 [nouR] transformants were obtained and checked for the restoration of growth on acetate [ace +]. 14 of them were [ace +], showing that wild-type GUP1 complemented ΔGUP1 mutation. Two of these complemented transformants were crossed with the wild-type S strain and [phleoR, nouR] ΔGUP1 pGUP1 as well as [phleoS, nouR] ΔGUP1 pGUP1 homokaryotic mat+ and mat-strains were purified.

Plasmid construction for GUN1- and GUN1^SG-mCherry/mCherry-AKI tagging

Two kinds of tagging were undertaken: one with the mCherry-AKI reporter protein, carrying the AKI PTS1 peroxisome targeting signal present in C-terminus of GUN1, added in C-terminus of the mCherry, and the second with the standard mCherry without the AKI PTS1 signal. To this end, we constructed plasmids allowing integration of the mCherry-AKI CDS or the mCherry CDS in 3’ (and in frame) of GUN1 or of GUN1^SG CDS at the endogenous GUN1 locus by homologous recombination (Figure S7). To achieve this, the 621 bp region upstream of the stop codon (but downstream of the mutation present in the GUN1^SG allele) was PCR-amplified with primers 1340GFP_F2 and 1340GFP_R1 (Table S2) designed to incorporate the ApaI and XhoI restriction sites in the sequence respectively. The PCR product was cloned blunt-end into the pBC-genR plasmid previously digested with EcoRV. The insert was then sequenced, digested with the restriction enzymes ApaI and XhoI and gel purified to be finally cloned upstream to the mCherry CDS into the pBC-mCherry-hygR plasmid digested by ApaI and XhoI (Table S2). This pBC-GUN1-mCherry-hygR (renamed pGUN1-mCherry for sake of simplicity) plasmid was sequenced and transformed into both Δmus52::genR and GUN1^SG Δmus52::genR and [hygR] transformants were selected. We obtained 1 [hygR] transformant for Δmus52::genR and 2 [hygR] transformants for GUN1^SG Δmus52::genR. Every transformant showed red fluorescence under the microscope. Correct GUN1-mCherry and GUN1^SG-mCherry gene fusions were verified by sequencing (data not shown). One transformant of each genotype was selected and crossed with the S strain to purify [hygR, genS] GUN1-mCherry and GUN1^SG-mCherry homokaryotic mat- and mat+ strains in the progeny. Finally, we observed that GUN1-mCherry ascospores germinated as the wild-type and that GUN1^SG-mCherry ascospores germinated spontaneously on M2 medium. To construct the pBC-GUN1-mCherry-AKI-nouR plasmid, we amplified by PCR the insert present in pGUN1-mCherry with primers 1340GFP_F2 and mCH_AKIR1, the latter primer allowing addition of the AKI coding sequence at the end of the mCHerrry (Figure S7). This 1341 bp PCR fragment was cloned blunt-end into pBC-nouR digested with EcoRV to give the pBC-GUN1-mCherry-AKI-nouR plasmid (renamed pGUN1-mCherry-AKI). The construction was sequenced and transformed into the Δmus52::genR strain. We selected one [nouR] transformant showing red fluorescence under the microscope and we crossed it with the wild-type S strain to purify [genS, nouR] GUN1-mCHerry-AKI mat+ and mat-strains. This GUN1-mCHerry-AKI strain showed red fluorescence under the microscope and correct GUN1-mCHerry-AKI gene fusion was verified by sequencing (data not shown). This GUN1-mCherry-AKI strain was then crossed with the GUN1^SG mutant to generate the GUN1^SG-mCherry-AKI strain by meiotic recombination between the GUN1^SG mutation and the mCherry-AKI-nouR insertion. In the progeny of this cross, we isolated 13 thalli from spontaneously germinating [nouR] ascospores on M2 medium supplemented with Nourseothricin. All these isolates were heterokaryotic auto-fertile (mat+ / mat-). Since the GUN1^SG mutation is recessive, we hypothesized that these isolates arose from GUN1^SG-mCherry-AKI/GUN1^SG-mCherry-AKI recombinated heterokaryotic ascospores. One of these isolates was fragmented and [NouR] homokaryotic GUN1^SG-mCherry-AKI ascospores of each mating type were purified establishing the GUN1^SG-mCherry-AKI strain. Red fluorescence in this final strain was verified and the presence of the GUN1^SG mutation as well as correct mCherry-AKI integration were verified by sequencing (data not shown). Germination of GUN1^SG-mCherry-AKI ascospores was spontaneous on M2 medium as for the GUN1^SG mutant.

Mycelium fragmentation and strain purification

For strains carrying the ΔGUN1 deletion and which therefore cannot germinate, homozygous crosses were performed on M2 medium supplemented with Tricyclazole (1 μg.mL^-1), leading to spontaneous germination of ascospores. The purification of homokaryotic strains was performed through mycelium fragmentation as follows: a small implant of the thallus of interest (0.5 cm²) was set in 2 mL tube containing 500 μL H₂O and ground using a FastPrep (TeSeE - Biorad, Hercules, California, United States) (20s, 5000 rpm). 100 μL of the fragmented mycelium were then spread on an agar plate, and small hyphal fragments were isolated, using a binocular (magnification x40). These isolates were then placed on M2 medium and cultured for 2 days at 27°C. Homokaryotic (mat+ or mat-) versus auto-fertile heterokaryotic (mat+/mat-) genotype of isolates was determined through a mat-type test using wild-type S mat+ and mat- strains.

Fertility assay

Fertility was assayed by generating auto-fertile mat+/mat– heterokaryons of the tested strains. To this end, implants (0.5 cm²) of each mating type of the strains of interest were placed in 2 mL tubes containing 500 μL H₂O and ground using a FastPrep (TeSeE - Biorad, Hercules, California, United States) (20s, 5000 rpm). 10 μL of each heterokaryon were dropped onto M2 medium and cultured for 10 days at 27 °C with light. A qualitative evaluation of perithecia formation directly on the plates and of ascospore production projected on the Petri plate lids during 4 days was performed. Every heterokaryon were analyzed in duplicate.

Cellophane penetration assay

An implant of each tested strain was placed on a cellophane layer (Biorad, Hercules, California, United States). After 2-, 3-, 4-, and 5-days growth at 27°C, the cellophane layer was removed, and the presence of mycelium in the medium checked with a binocular to determine if the strain had breached the cellophane layer. In parallele, the presence/absence and the morphology of appressoria in the cellophane layer were observed under the microscope as described in (Brun et al., 2009; Demoor et al., 2019).

Microscopic observations

Ascospores observations were performed in Ibidi 8-wells chamber micro-slides (Gräfelfing, Germany). Each well was filled with 200 μL of either M2 or modified G liquid medium: the quantity of Bacto peptone was divided by two compared to standard G medium in order to reduce the fluorescing background noise due to Bacto peptone. Germination induction of this modified medium was not altered (data not shown). Crosses were performed on standard M2 medium. Once perithecia were mature, agar plugs bearing the perithecia were cut and placed above the microscopic chambers upside-down. Perithecia were left to project their ascospores into the well for 5 hours. For mycelium observations, small squares of medium (1 cm²) with grown mycelium on it were cut at the edge of the thallus and placed upside-down in water on a coverslip of a microscopic chamber. Images were taken with an inverted microscope Zeiss spinning disk CSU-X1 (Oberkochen, Germany), with 4 lasers (405nm, 488nm, 561nm, 640nm) for fluorescence observations and their associated filters and a sCMOS PRIME95 (Photometrics) camera at the Imagoseine Imaging Facility: https://imagoseine.ijm.fr/676/accueil.htm. The images were analysed with Fiji (Schindelin et al., 2012).

Phylogenetic analysis

Fungal genes homologous to GUN1 were searched by BLAST in GenBanK and Mycocosm (Benson et al., 2013; Grigoriev et al., 2014), using the default parameters with the GUN1 protein sequence as query. For a selection of Ascomycota, Basidiomycota and Mucoromycota species, hits with an e-value lower than 10⁻⁵ were selected. Research for other homologs was carried out for each selected species on OrthoDB (Kriventseva et al., 2019). The alignment was performed with MAFFT (Katoh et al., 2005) and manually refined using Jalview (Waterhouse et al., 2009). The phylogenetic tree was built using the maximum likelihood method (PhyML software using the default parameters) (Guindon and Gascuel, 2003). The tree was visualized on the iTOL server (Letunic and Bork, 2007). Bootstrap values are expressed as percentages of 100 replicates (Figure S4). The GUN1 I441 residue conservation was assessed by visualizing the alignment on Jalview (Figure S6) (Waterhouse et al., 2009).

Bioinformatic analysis of the GUN1 protein

The analysis of the GUN1 protein sequence was carried out using multiple tools: CD-search: https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (Marchler-Bauer and Bryant, 2004), Interproscan: https://www.ebi.ac.uk/interpro/search/sequence/ (Quevillon et al., 2005) and Prosite: https://prosite.expasy.org/ (Sigrist et al., 2010). Prediction of its localization was realized using wolfPSORT: https://wolfpsort.hgc.jp/ (Horton et al., 2007). For research of a Mitochondrial Targeting Signal (MTS), we also used MitoFates: http://mitf.cbrc.jp/MitoFates/cgi-bin/top.cgi (Fukasawa et al., 2015). 3D homology modelling was performed using Swiss model: https://swissmodel.expasy.org/ (Waterhouse et al., 2018), and a 3D structure prediction of the GUN1 protein was achieved using the I-TASSER platform available at: https://zhanglab.ccmb.med.umich.edu/I-TASSER/ (Roy et al., 2010). The predicted effect of the missense I447N mutation in GUN1^SG on protein structure and stability was assessed using the STRUM server (https://zhanglab.ccmb.med.umich.edu/STRUM/) and the PROVEAN online tool (http://provean.jcvi.org/index.php) respectively (Choi and Chan, 2015; Quan et al., 2016).

CAT activity assay

Mycelium proteins extraction: Mycelia growing on M2 medium (2 confluent plates per strain) were harvested after 2 days, and placed in 2 mL tubes each one containing a tungsten bead (diameter 3 mm), 1 mL of potassium phosphate buffer (pH=7.4) supplemented with EDTA 2 mM and ground in a TissueLyser II apparatus (Qiagene, Hilden, Germany) at 30rpm/s for 4 min at 4°C. The lysate was centrifugated at 4°C for 20 min at 17000 g and the supernatant was separated from the pellet (cell debris). The protein concentration in the supernatant was measured by the spectrophotometric Bradford method (Sigma Chemical Co., St Louis). Ascospore proteins extraction: each strain was crossed (in homozygous crossing) on at least 5 M2 medium plates. When perithecia were mature, the projected ascospores were collected on agar plates topped with a cellophane layer and pulled together in a 2 mL tube for each strain, each one containing a tungsten bead (diameter 3 mm). Immediately after harvesting, each tube was flash frozen in liquid nitrogen. Ascospores were dry-crushed in a TissueLyser II apparatus (Qiagene, Hilden, Germany) at 30 rpm/s for 4 min at -80°C. Ascospores were maintained at -80°C in precooled TissueLyser blocks. The crushed ascospores were resuspended in 200 μL of potassium phosphate buffer (pH=7.4) supplemented with EDTA 2 mM. The lysate was centrifugated at 4°C for 20 min at 17000 g and the supernatant was separated from the pellet (cell debris). The protein concentration in the supernatant was estimated by the spectrophotometric Bradford method (Sigma Chemical Co., St Louis). Carnitine-acetyltransferase assay: Carnitine-acetyltransferase (CAT) activity was assayed as described in (Kawamoto et al., 1978). The reaction was monitored spectrophotometrically at room temperature by following the release of CoA-SH from acetyl-CoA using the thiol reagent 5, 5’-dithiobis-nitrobenzoic acid (DTNB-Sigma Chemical Co., St Louis). The reaction mixture contained 100 mM Tris-HCl buffer (pH 7.8) 0.05 mM acetyl-CoA (Sigma Chemical Co., St Louis), 0.1 mM DTNB, 22 mM DL-carnitine chloride (Sigma Chemical Co., St Louis) and protein extract in final volume I.0 mL. The reaction was initiated by adding a volume of the protein extract and the increase in absorbance was followed at 412 nm. CAT activities were determined by measuring initial velocity of the CoA-SH production reaction, and then reported as the activity ratio of the wild-type S strain. Standard deviations and statistical analyses were calculated on 4 to 7 biological replicates. Eventually, CAT activities were compared using a Fisher-Pitman Permutation Test.

Western Blot analysis

PaMpk2 phosphorylation was assessed as described in (Lalucque et al., 2012). Ascospores produced by homozygous crosses of the S strain, the PaMKK2^c mutant and the GUN1^SG mutant were harvested on agar plates topped with a cellophane layer to facilitate the ascospores harvesting process. For germination induction, a cellophane layer with wild-type ascospores was transferred on G+YE medium for 2 hours. Once collected, ascospores were flash frozen in liquid nitrogen, then dry-disrupted in a Micro-Dismembrator (Sartorius) at 2600 rpm for 1 min at -80°C. Crushed ascospores were resuspended in Laemli buffer, placed 5 minutes at 100 °C, then centrifuged for 15 min at 14000 rpm. Samples were placed on a 15% SDS-PAGE gel 1 mm thick, and migrated for 3 h at 130V, 25mA/gel and 25W. The gel was then transferred onto a PVDF membrane. Hybridization with the anti p44/p42, or anti-phospho p44/42 (Cell Signaling Technology) antibodies, diluted to 1/1000, was carried out overnight at 4°C. Hybridization with the second antibody coupled to peroxidase (GEHealthcare), diluted to 1/1000, was carried out for 1 h at room temperature. For the revelation, the Immobilion® chemiluminescence kit (Millipore) was used according to the supplier’s recommendations.

Authors contribution

AD: experimentation and manuscript writing; IL: experimentation; RF: experimentation; CL: statistics analyses; PS: project initiator: isolation & genetic analysis of the SGD mutants, manuscript writing; SB: experimentation, experiments conceptualization, manuscript writing, project leader.

Funding

This work was funded by Université Paris Cité intramural funding. Genome sequencing was made possible by grant PSUD/SAIC N°9283-0 to P. Silar. S. Brun was supported by IdEx Université Paris Cité: ANR-18-IDEX-0001

Conflict of interest

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

Acknowledgments

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 »). We also thank Sylvie Cangemi for her precious help in preparing the various media used in this study and Elizabeth Guinot Bordini for English proofreading.