Mutations in Mating-Type Genes of the Heterothallic Fungus Podospora anserina Lead to Self-Fertility

Corresponding author: Sylvie Arnaise, Institut de Génétique et Microbiologie, Bâtiment 400, Université Paris-Sud, F-91405 Orsay Cedex, France., arnaise{at}igmors.u-psud.fr (E-mail)

Communicating editor: M. E. ZOLAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The heterothallic fungus Podospora anserina has two mating-type alleles termed mat+ and mat-. The mat+ sequence contains one gene, FPR1, while mat- contains three genes: FMR1, SMR1, and SMR2. FPR1 and FMR1 are required for fertilization, which is followed by mitotic divisions of the two parental nuclei inside the female organ. This leads to the formation of plurinucleate cells containing a mixture of parental mat+ and mat- nuclei. Further development requires a recognition between mat+ and mat- nuclei before migration of the mat+/mat- pairs into specialized hyphae in which karyogamy, meiosis, and ascospore formation take place. FPR1, FMR1, and SMR2 control this internuclear recognition step. Initial development of the dikaryotic stage is supposed to require SMR1; disruption of SMR1 results in barren perithecia. In a systematic search for suppressors restoring fertility, we isolated 15 suppressors—all of them mutations in the mating-type genes. These fmr1, smr2, and fpr1 mutants, as well as the strains disrupted for FMR1, SMR2, and FPR1, are weakly self-fertile. They are able to act as the male partner on a strain of the same mating type and give a mixture of biparental and uniparental progeny when crossed with a wild-type strain of opposite mating type. These observations lead us to propose that SMR2, FMR1, and FPR1 act as activators and repressors of fertilization and internuclear recognition functions.

THE mating-type alleles, which were defined as controlling fertilization, appear to control some additional events after fertilization in the fungus Podospora anserina (ZICKLER et al. 1995 ). In heterothallic filamentous ascomycetes such as P. anserina, in which each nucleus contains a single copy of one of two mutually exclusive mating-type loci, sexual reproduction begins with recognition between a female organ (the ascogonium) and a male cell (the microconidium) of opposite mating type. This recognition event leads to fertilization, during which the male nucleus is imported into the ascogonium. However, karyogamy between nuclei of opposite mating type does not take place immediately. Instead, the male nucleus undergoes several mitotic divisions resulting in the formation, inside the female organ, of plurinucleate cells containing both male and female nuclei. Therefore, nuclei of male and female origin (of opposite mating type) must recognize each other before they are isolated within the cell that gives rise to the ascus. This internuclear recognition, called IR (DEBUCHY 1999 ), is thus also associated with a transition from a syncytial stage to a cellular stage, which requires that the two nuclei of opposite mating types migrate from the syncytial cell into a specialized hypha (the ascogenous hypha) where they divide mitotically, maintaining a strict ratio of 1:1 of each parental nucleus. Eventually pairs of nuclei fuse and meiosis ensues immediately, resulting in the expected Mendelian ratio of each mating-type allele in the progeny.

The mating-type locus consists of two exclusive alleles, mat+ and mat-. The mat+ sequence contains a sole gene, FPR1, and the mat- sequence contains three genes: FMR1, SMR1, and SMR2 (DEBUCHY and COPPIN 1992 ; DEBUCHY et al. 1993 ). These genes, except SMR1, encode regulatory proteins related to two well-known transcription factor families, the HMG family and the proteins related to MAT1 of Saccharomyces cerevisiae. SMR1 does not display any functionally characterized motif; however, it contains a highly conserved region also present in matA-2 of Neurospora crassa (FERREIRA et al. 1996 ) and SmatA-2 of Sordaria macrospora (POGGELER et al. 1997 ). This region has been proposed to define a new family of transcription factors (DEBUCHY et al. 1993 ).

Mutations in the C terminus of FMR1 or SMR2 lead to the formation of uninucleate ascogenous hyphae and progeny in which the mat+ parent is absent. This phenotype has been interpreted as resulting from an altered property of mutant mat- nuclei, which become able to proceed alone through the developmental steps that are possible only for two compatible nuclei in the wild-type strains. It was suggested that wild-type FMR1 and SMR2 ensure that mat- nuclei express a property required for their recognition by mat+ nuclei (ZICKLER et al. 1995 ; ARNAISE et al. 1997 ). This property has been termed nuclear identity. However, the nature of the nuclear identity alteration observed in fmr1 or smr2 mutants, as well as the reasons for uninucleate ascogenous hypha formation, remain unclear. Similarly, fpr1 mutants that have conserved the fertilization domain lead to selfish mat+ mutant nuclei equivalent to the selfish mat- mutant nuclei (ZICKLER et al. 1995 ). Disruption of SMR1 does not prevent the mutant strain from crossing with a mat+ wild-type strain, but perithecia are barren and no dikaryotic specialized hyphae are formed. It has been proposed that SMR1 is required for the recovery from a developmental arrest resulting from IR (COPPIN and DEBUCHY 2000 ).

We have undertaken a systematic search for suppressor genes allowing the formation of progeny in a cross between a disrupted SMR1 strain and a mat+ strain. Such mutations should permit the by-pass of the requirement for SMR1. Surprisingly, the 15 mutants obtained mapped in FMR1, SMR2, or FPR1. All mutants show developmental phenotypes similar to those previously described for the mat mutants, but they are self-fertile, a feature that proved to have been overlooked in former mat mutants. This suggests that the wild-type mat- genes FMR1 and SMR2 are required for the repression of mat+ functions in addition to being necessary for the expression of mat- functions, whereas FPR1 has converse effects on mat+ and mat- functions. On the basis of these dual functions, we propose a model for the selfish behavior of mat mutant nuclei.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

P. anserina culture and genetic analysis:
The characteristics of P. anserina were first described by RIZET and ENGELMANN 1949 and then reviewed by ESSER 1974 . Most asci contain four binucleate ascospores. A few asci contain five ascospores: three binucleate and two smaller uninucleate ascospores that produce homokaryons. Each binucleate ascospore is formed around two nonsister nuclei after a postmeiotic mitosis. Binucleate ascospores are generally self-fertile, because the two alleles that control mating types display a 98% segregation at the second division of meiosis. The 136 mutation, which prevents full ascospore and mycelium pigmentation, exhibits a first division segregation; 98% of the asci contain two green and two black ascospores (MARCOU and PICARD 1967 ). The uniparental progeny are composed of asci and of scattered ascospores containing markers from one parent only. For instance, in a cross between a mat- mutant strain and a mat+ 136 strain, uniparental mat- asci contain four black ascospores while biparental asci contain two black and two green ascospores. Uniparental scattered ascospores, which can be binucleate or uninucleate, are black. These scattered ascospores belong to asci that contain at least one viable ascospore while the others have undergone abortive development, most often because they contain aneuploid nuclei (ZICKLER et al. 1995 ). When the frequency of asci containing four black ascopores was <20% of the total progeny, a systematic analysis of the segregation of the mat marker was performed for 20–30 asci to determine if the asci containing four black ascospores were due to segregation at the second division of the 136 marker (which is recessive) or to uniparental progeny.

Crosses were performed by spermatization (spraying of microconidia onto strains of opposite mating type). When crosses were performed to separate male and female functions, the strains to be used as male or female partner were grown separately on petri dishes containing minimal medium, and the female partner was incubated at 27° in the light to allow formation of the female organs. On the sixth day of culture, microconidia of the strain to be used as the male partner were recovered with 2 ml of sterile water and spread on mycelia of the female partner strain.

Nomenclature:
To simplify the nomenclature, the disrupted genes FMR1::ura5, SMR1::ura5, and SMR2::ura5 were named fmr1-r, smr1-r, and smr2-r, respectively. The FPR1::ura5 disrupted gene (see below) was named fpr1-r for the same reason. All the new mutants obtained in FMR1, SMR1, SMR2, and FPR1 are lowercase. The wild-type ectopic copy of a gene in a strain is noted in uppercase.

mat mutant strains previously obtained:
Gene disruptions were introduced at the mat- idiomorph by homologous recombination and the resulting strains called smr1-r, smr2-r, and fmr1-r (ARNAISE et al. 1997 ). The fpr1-1 mutant strain was obtained by transformation of the mat^SK strain (see below) with a 1727-bp NcoI-NcoI fragment containing a truncated FPR1 gene coding for a polypeptide lacking the 121 C-terminal amino acids. This fpr1-1 mutant gene was integrated at an ectopic site (ZICKLER et al. 1995 ). In the fpr1-1 and fmr1-r mutants, the N-terminal part of the polypeptide necessary for fertilization was unchanged. The mat+ SMR1 SMR2 was obtained by transforming a mat+ strain with a plasmid containing both SMR1 and SMR2 sequences and the ble gene conferring resistance to phleomycin (ARNAISE et al. 1997 ).

Disruption of the FPR1 gene:
FPR1 was disrupted by insertion of the ura5 gene from P. anserina (TURCQ and BEGUERET 1987 ) downstream of the region necessary for fertilization. The plasmid puraN14EP with the disrupted FPR1 was used to transform a mat+ ura5-6 strain. Previous analyses of the fpr1-1 ectopic mutant showed that sporulation efficiency was affected when the mutant was crossed with a mat- strain (ZICKLER et al. 1995 ). Therefore, the sporulation phenotypes of 390 transformants were tested in crosses with a mat- strain. Primary [ura⁺] transformants were crossed with a mat- ura5-6 strain. A ura5-6 x ura5-6 cross leads to barren perithecia, while a ura⁺ x ura5-6 cross is fully fertile. Consequently, progeny can result only from the transformed nuclei. Ten transformants exhibited a decrease in sporulation efficiency. Since primary transformants contain both transformed and untransformed nuclei, they were purified by crossing with a tester strain of opposite mating type and by selecting homokaryotic transformed strains in the progeny. Crosses of these purified transformants with a mat- ura5-6 strain showed that the [ura⁺] phenotype segregated with the mat+ locus. Four of them were submitted to Southern blot analysis. Three displayed rearrangement in the FPR1 region and one was shown to have the expected disruption of the FPR1 gene. This latter transformant was named fpr1-r.

Construction of the mat+/smr1-r strain:
The mat+/smr1-r strain is a heterokaryotic strain issued from binucleate ascospores and therefore contains a mixture of mat+ and smr1-r nuclei. It was obtained from a cross between a mat+ SMR1 SMR2 strain and a smr1-r strain. SMR1 in the mat+ nucleus can complement the smr1-r disruption present in the partner and allows the production of progeny (ARNAISE et al. 1997 ). mat+/smr1-r strains were issued from binucleate ascospores that did not display resistance to phleomycin and thus did not carry ectopic SMR1 and SMR2 genes. The genotype of these binucleate ascospores was confirmed by crossing them with the mat- and the mat+ tester strains.

Selection of smr1-r revertants:
The heterokaryotic strain mat+/smr1-r differentiates perithecia that do not sporulate. To obtain sporulation-competent revertants, the mat+/smr1-r strain was inoculated on 3-cm cellophane discs plated on solid medium (M2) and grown for 2 days at 27°. The thalli were treated by ultraviolet irradiation at 100, 150, 200, and 300 J/m² (240, 120, 240, and 120 thalli, respectively). Eighty untreated thalli were reserved as a control for spontaneous reversion. The discs were then transferred onto new M2 dishes and put back in the light at 27°. After about 1 week, perithecia were formed and a systematic search for appearance of ascospores or asci was undertaken. Fifteen independent sporulation sectors were obtained. For each of them, all the scattered ascospores or the asci were picked up. A, B, and C strains correspond to different progeny from different mat+ SMR1 SMR2 x smr1-r crosses. Revertant strains were termed smr1-r su or mat+ su, according to the nucleus that contains the suppressor.

Construction of the strains used for complementation of suppressors:
To obtain the mat+ SMR1 strain, transformants resistant to hygromycin were recovered upon transformation of the wild-type mat+ strain with a pCBSMR1 plasmid containing SMR1 (see construction below). To determine whether the hygromycin-resistant transformants carried a functional SMR1, they were crossed with a smr1-r strain. Sterility of this strain is complemented by a functional SMR1 either in the mat- or in the mat+ partner.

The mat+ SMR2 strain was obtained by transforming a mat+ strain with the plasmid pCBSMR2, containing SMR2 (COPPIN and DEBUCHY 2000 ).

The mat+ FMR1 strain was selected as a hygromycin-resistant transformant upon transformation of the mat+ strain with the pCBFMR1 plasmid that contains FMR1 (see construction below). Introduction of a FMR1 transgene into a mat+ strain induces self-fertilization. The postfertilization function was examined by introgressing the ectopic FMR1 gene in a fmr1-r strain.

The mat- FPR1 strain was obtained by crossing a mat^SK R1R5 strain, in which the R1R5 fragment carries FPR1, with a mat- strain. The mat^SK R1R5 itself was obtained through the following steps: the mat^SK strain is a strain deleted from a large part of the mat+ specific sequences (COPPIN et al. 1993 ). A leu1-1 mat^SK strain was transformed with the pHMTPP plasmid containing the whole mat- information and the su8-1 tRNA gene that encodes a leu1-1 suppressor (DEBUCHY and COPPIN 1992 ). The leu1⁺ mat- resulting strain was then cotransformed with the KSR1R5 plasmid (containing the entire FPR1 gene) and the pUT703 plasmid containing the ble gene that confers phleomycin resistance. This transformant was crossed with a mat- strain and a mat^SK R1R5 was obtained among their progeny.

Construction of the strains used for selfing test:
The pah1 mutation is a deletion in the homeobox gene pah1 (ARNAISE et al. 2001 ). The nature of the IncA mutation is unknown but this mutation confers a female sterile and a "super male" phenotype (MARCOU et al. 1993 and reference therein). The pah1 smr1-r fmr1^95-107 SMR1 SMR2 strain was obtained by a cross between smr1-r fmr1^95-107 FMR1 and mat+ pah1 SMR1 SMR2. The SMR1 SMR2 sequence, integrated at an ectopic position (see above), and the pah1 gene segregated independently of the mat locus. The smr1-r fmr1^95-107 FMR1 strain was obtained by transforming the smr1-r fmr1^95-107 (R10) strain with the pCBFMR1 plasmid (which contains FMR1). The IncA mat and the pah1 mat strains were obtained by a cross between a mat^SK R1R5 (see above) and either the IncA or the pah1 strain of opposite mating type.

Counting of ascospores:
The entire progeny issued from a cross on a petri dish was recovered on its lid. For an ascospore density <50,000 per lid, the ascospores present on a quarter of the lid surface were counted and this number was multiplied by four to obtain the total progeny of the cross. For lids containing >100,000 ascospores, the progeny were estimated by visual comparison with two reference lids chosen for their difference in ascospore density: the ascospores present on an eighth of the lid surface were counted and this number was multiplied by eight to obtain the total progeny present on each lid.

Counting of microconidia and perithecia:
The relevant strains were grown on petri dishes containing minimal synthetic medium (M2) and incubated at 27° in the dark. The microconidia were recovered after 6 days of culture by washing the surface of the mycelia with 2 ml of sterile water. This allowed us to recover 1 ml of microconidia suspension, which was counted under the microscope in a hematimeter chamber. To test their fertilization ability, 1 ml of microconidia suspension (after dilutions) was spread on wild-type mycelia used as female partners, which were previously grown on M2 medium at 27° during 6 days in the light to allow formation of female organs. Perithecia were counted 5 days after fertilization.

Light microscopy preparations:
These were performed as previously described by ZICKLER et al. 1995 .

Bacterial strains, plasmids, and plasmid constructions:
Cloning and plasmid preparations were done in either Escherichia coli HB101 (BOYER and ROULLAND-DUSSOIX 1969 ) or DH5 (HANAHAN 1983 ).

pCBSMR1 is based on pCB1004 (CARROLL et al. 1994 ) and contains SMR1 on a 2.1-kb AseI-EcoRI fragment derived from pULP68 (COPPIN and DEBUCHY 2000 ). pCBSMR2 is based on pCB1004 and contains SMR2 on a 2.5-kb EcoRI-PstI fragment derived from pULP (COPPIN and DEBUCHY 2000 ). pCBFMR1 is based on pCB1004 and contains FMR1 on a 2.3-kb EcoRI-XbaI fragment derived from pULP (DEBUCHY et al. 1993 ).

Plasmid puraN14EP contains the FPR1 gene with a disruption downstream of the region encoding the HMG domain. It is based on pucEP, which contains the 20-kb EcoRI-PstI fragment encompassing the mat+ idiomorph (DEBUCHY and COPPIN 1992 ). Plasmid pucEP has been partially digested with NcoI, molecules with one cut have been isolated on an agarose gel, treated with the Klenow enzyme, and ligated with a 1.6-kb blunt-end fragment containing the ura5 gene. This blunt-end fragment was obtained by S1 digestion of the 1.6-kb EcoRI fragment of pPAura5-1 (TURCQ and BEGUERET 1987 ). Restriction digests of recombinant plasmids allow us to identify insertion of the ura5 gene in the NcoI site of pucEP corresponding to residue 282 of FPR1.

The plasmid KSR1R5 is a Bluescript derivative carrying the 4.1-kb EcoRI-EcoRV fragment of the mat+ locus containing the whole FPR1 gene (DEBUCHY and COPPIN 1992 ).

DNA procedures and sequencing:
Genomic DNA was prepared using the rapid petri dish-grown mycelia method (LECELLIER and SILAR 1994 ). The SMR2 gene from the 11 revertants from class 2 was amplified using polymerase chain reaction (PCR) with the pair of primers, 278028 (5'-GATATTATTCTGCCACTCCC-3') and 1884 (5'-CTGAACCAACGTCTGGTGC-3'). The FPR1 gene from the three revertants of class 3 was amplified by PCR with the pair of primers, E1 (5'-TCAATCTCAGCATCCGAGAC-3') and F13 (5'-GCGGAAGTGATCAGAATTGA-3'). The FMR1 gene from the unique revertant of class 1 was amplified by PCR with the pair of primers, 765 (5'-GTTTGCCTTCATTTCATCCC-3') and 2526 (5'-GACCTCCCGCCCTCGGTCGG-3'). The amplification products were then sequenced using the ABI PRISM Ready Reaction DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA), with an automatic sequencing machine (373 A DNA sequencer; Applied Biosystems).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Revertants of the smr1-r developmental arrest belong to three different classes of mating-type phenotypes:
smr1-r corresponds to a disruption of the coding sequence of the SMR1 gene leading to barren perithecia in a smr1-r and mat+ strain cross. The heterokaryotic strain smr1-r/mat+ was used to select revertants able to sporulate, as described in MATERIALS AND METHODS, and 15 sporulating sectors were obtained after mutagenesis. For each sector, all the ascospores that had germinated (1–27) were crossed by mat- and a mat+ tester strains. This allowed us to group the revertants into three classes according to their mating-type phenotype. In the first class, all ascospores of a given sector were unable to mate with strains of either mating type (1 revertant: R10). In the second class all ascospores were mat- (11 revertants: R1–R7 and R11–R14) and in the third class all ascospores were mat+ (3 revertants: R8, R9, and R15). Each sporulating sector was considered to be the progeny of one mutation event and only one ascospore of each sporulating sector was analyzed further.

The suppressor of the first class resulted from a mutation in the FMR1 gene:
To characterize the molecular event leading to the inability to mate with the mat- or mat+ tester strains, the structure of the mating-type locus of the R10 strain was determined by PCR analysis. The two pairs of primers specific for the FPR1 gene gave no amplification product, but an amplification product smaller than expected was obtained with the two pairs of primers specific for the FMR1 gene. Sequencing of this amplification product revealed a deletion of 31 bp, 282 bp downstream of the ATG in the FMR1 gene. This mutation corresponded to an in-frame deletion of 13 amino acids (95–107) just after the 1 domain (Fig 1); it was named fmr1^95-107 and its corresponding strain, smr1-r fmr1^95-107. A wild-type copy of the FMR1 gene, introduced by transformation, can complement the fertilization defect of the smr1-r fmr1^95-107 strain. This confirms that the mutant phenotype of the R10 strain was caused by the mutation in the FMR1 gene and indicates that the fmr1^95-107 mutation is recessive.

View larger version (14K): In this window In a new window Download PPT slide   Figure 1. SMR1, FMR1, SMR2, and FPR1 protein structures and positions of mutations for each revertant. Triangle, position of the ura5 gene insertion. Shaded box, amphipathic helix; black dotted box, conserved region between SMR1 and SMATA-1 of S. macrospora and MATA-1 of N. crassa; diagonally hatched box, HMG boxes; dotted box, 1 motif; thin vertical bars, position of the mutated residues in the revertants; thick vertical bars, amino acid numbers; open box, deletion fmr195-107 in the R10 revertant; asterisk (*), stop; thick horizontal bars, N-terminal portions of FPR1 and FMR1 required for fertilization. Amino acids are numbered from the putative ATG.

The suppressors of the second class resulted from mutations in the SMR2 gene:
The revertants with the mat- mating type were crossed with the 136 mat+ tester strain. The 136 mutation is a spore color marker allowing easy detection of uniparental progeny (see MATERIALS AND METHODS). The sporulation efficiency was decreased compared to a wild-type cross and the progeny was exclusively uniparental mat-. To ascertain that the reversion events were not a loss of the insertion of the ura5 gene that disrupts the SMR1 gene, PCR experiments were performed with two primers in the SMR1 gene flanking the ura5 gene. In all the revertants we amplified a fragment of the expected size for an intact insertion of the ura5 gene. Thus, all the revertants had the smr1-r mutation and an extragenic suppressor mutation (su) in their mat- nucleus. SMR1 can fulfill its function whatever its location, in either a mat- or a mat+ nucleus (ARNAISE et al. 1997 ). To determine the phenotype of the suppressors, we complemented the SMR1 disruption by crossing each revertant (smr1-r su) by a mat+ 136 strain with an ectopic SMR1 gene. All the suppressors led to mat- uniparental and biparental progeny (Table 1). For the suppressors R2, R11, and R13 the biparental progeny are quantitatively comparable to those of a wild-type cross. However, these suppressors also gave 0.3–6% uniparental progeny, a feature never observed in a wild-type cross (10⁶ asci were analyzed; S. ARNAISE, unpublished data). For the other suppressors the progeny were decreased (one-tenth of the wild-type progeny) and the percentage of uniparental progeny was 23–99%. To localize the suppressors, the biparental progeny of the smr1-r su x mat+ SMR1 cross were further analyzed. Depending on the revertant, 8–86 mat- ascospores, which had not received the wild-type SMR1 gene from the mat+ SMR1 parent, were crossed with a mat+ tester strain. These crosses produced only uniparental mat- progeny, as observed in the initial test cross of the revertant. Thus, for all the revertants, the suppressor phenotype cosegregated with mat-.

Since these suppressors displayed phenotypes similar to those of smr2 or fmr1 mutants, and were close to the mat- locus, we then tested if they could be alleles of SMR2 or FMR1. We therefore constructed smr1-r su SMR2 and smr1-r su FMR1 strains, introducing by crosses a SMR2 or a FMR1 ectopic gene in each revertant strain. Because smr1-r su strains give only uniparental progeny in crosses with a mat+ strain, smr1-r must first be complemented by SMR1 to allow segregation of FMR1 or SMR2 from the mat+ strain with the suppressor of the revertant strains. smr1-r su SMR1 strains were obtained from a cross between smr1-r su and mat+ SMR1. SMR2 or FMR1 transgenes were then associated with the mutant mat- idiomorph by crossing the smr1-r su SMR1 strains with a mat+ SMR2 or a mat+ FMR1 strain.

All the smr1-r su SMR2 strains displayed the phenotype of a smr1-r strain (sterile), while the smr1-r su FMR1 strains displayed the phenotype of a smr1-r su strain (uniparental progeny). Thus, in all the revertants the wild-type SMR2 gene complemented the suppressor, in contrast to FMR1. Sequencing of the SMR2 gene in each revertant revealed either missense (R1, R2, R3, R3, R4, R5, R6, R11, R13, and R14) or nonsense (R12) mutations in SMR2 (Table 1 and Fig 1), except for the R7 mutant, which resulted from a mutation in the first base of the second intron (smr2³⁹⁸). The R3 and R6, R4 and R14, and R5 and R11 carry the same mutations. These mutations could not preexist in the strain before mutagenesis because, for each pair of revertants, the mutagenized strain was different (Table 1). Among the eight different SMR2 mutations five were localized in the HMG domain of the protein and two upstream of this domain. Each revertant was further named using the number of the mutated residue (for example, R1 was named smr1-r smr2^I26T).

The suppressors of the third class resulted from mutations in the FPR1 gene:
Since the revertants of the third class (R8, R9, and R15) were mat+, they were crossed with a smr1-r strain to determine if they contained a suppressor mutation. These crosses gave an exclusively uniparental mat+ progeny, confirming the presence of a suppressor mutation in the mat+ nucleus. All these suppressors led to both mat+ uniparental progeny and biparental progeny when crossed to a mat- 136 strain. Except for R8, the uniparental progeny represented <1% of the total progeny (Table 2). To localize the suppressor mutations each mat+ su revertant was crossed with a smr1-r SMR1 strain and we analyzed 15 mat- ascospores that had not received the wild-type SMR1 gene from the smr1-r SMR1 parent. In all cases, all mat– progeny displayed the smr1-r phenotype (sterile) upon a cross with a mat+ tester strain. Thus the suppressor mutations are genetically linked to the mat+ locus. To determine if they were alleles of the FPR1 gene, we made the mat+ su FPR1 strains by crossing the mat+ su strains with the mat- FPR1 strain. The mat+ su FPR1 strains were unable to produce progeny when crossed with a smr1-r strain. Thus, for the three revertants, a wild-type FPR1 gene complemented the suppressors. Sequencing of the FPR1 gene in each revertant revealed missense mutations in FPR1, one in the HMG domain of the protein and two upstream of this domain (Table 2 and Fig 1). The three mutations are thus localized in the N-terminal portion, previously described as necessary for fertilization. However, the fertilization event did not seem affected in the three mutants as shown by their male fertility test (data not shown).

The fmr1, smr2, and fpr1 mutant strains are self-fertile:
All fmr1, smr2, and fpr1 mutant strains obtained from the search for smr1-r revertants are self-fertile (Table 3). This self-fertility was detected primarily when the mating-type mutation was associated with the pah1 mutation for the fpr1^T66I strains (R15). The pah1 mutation is a loss of function of the homeobox gene pah1, which is a repressor of microconidiogenesis (ARNAISE et al. 2001 ). The pah1 mutant strain produces 40 times more microconidia than a wild-type strain, suggesting that the self-fertility is increased when the number of microconidia is enhanced. This prompted us to reexamine the previously described fmr1-r and smr2-r phenotypes (ARNAISE et al. 1997 ) and to test the fpr1-r disruption. In fact, these mutants are also self-fertile. Their selfing is weak, as only 1–300 perithecia are formed per petri dish, compared to the 5000 perithecia seen in a wild-type cross made in the same condition. The number of ascospores produced is also very low; a maximum of 100 ascospores are produced, while a wild-type cross produces at least 100,000 ascospores per petri dish. In the case of smr1-r fmr1^95-107 (R10), self-fertility can be detected only when the mating-type mutation is associated with the pah1 mutation. In fact, self-fertility is enhanced when the mating-type mutation is associated with the pah1 mutation in fpr1^T66I (R15), fmr1-r, and smr2-r. All ascospores produced by selfing have the phenotype of the strain from which they issued, indicating that no cross contamination has occurred. The germination frequency of these ascospores is low (35–85%) as compared to the near 100% of the wild-type ascospores. The fmr1^95-107 or smr2 mutations are associated with the smr1-r mutation; therefore, we cannot separate them by crossing. To differentiate the phenotype of the revertants from the phenotype of the suppressors, we introduced a wild-type copy of the SMR1 gene in the revertants by crossing. No differences in the self-fertility phenotype were observed between the revertant strains (smr1-r smr2 or smr1-r fmr1^95-107) and the strains complemented for SMR1 (suppressor alone strains; data not shown).

All the fmr1, smr2, and fpr1 mutant strains can act as male partners on a strain of the same mating type:
We have tested whether the self-fertile mutant strains can fertilize a wild-type strain of the same mating type. The results of these crosses are shown in Table 4 and Table 5. No fertilization can be detected when the single mating-type mutants are used. When the mating-type mutations are associated either with the pah1 mutation or with the IncA mutation, fertilization events can be detected. The IncA mutant produces 1000 times more microconidia than wild type. The role of the IncA gene is unknown. It is likely that, in both IncA and pah1 mat mutant strains, the increase in the number of fertilization events is a consequence of the increase of the number of microconidia. This hypothesis is supported by the correlation between the number of microconidia that are produced by pah1 and IncA mutant strains and the increase observed in the number of perithecia resulting from the association of either mutation with mating-type mutations. This assumption was verified by diluting microconidia of a pah1 fmr1-r strain before crossing with a mat- strain. Fertility decreases with the number of microconidia (data not shown). In all the crosses of smr2 or fmr1 with mat- or fpr1 with mat+, all the progeny have the phenotype of the mating-type mutant parent. This indicates that the mating-type mutant nucleus is able to proceed through the developmental steps leading to ascospore formation.

A wild-type copy of the SMR2 gene, introduced by crossing in the smr2-r mutant, complemented the two phenotypes: selfing and crossing with a strain of the same mating type. This smr2-r SMR2 strain is incapable of selfing or of crossing with a mat- strain (data not shown).

Self-fertile perithecia contain mainly uninucleated croziers and exhibit haploid meioses:
Although rare, the perithecia formed by selfing of smr2, fmr1, and fpr1 are similar to those of wild type in morphology, but they remain mainly barren. Wild-type perithecia develop after fertilization of a female reproductive organ by a male nucleus carrying a nucleus whose mating type differs from that of the female. The female organ is plurinucleate and both male and female nuclei undergo several mitotic divisions in a common cytoplasm before a pair of nuclei of opposite mating type are isolated in a crozier cell. Thus, these nuclei must recognize their partners among many others to form a correct pair. Partitioning of nuclei in the crozier is followed by synchronous mitosis and two daughter nuclei of opposite mating type are isolated in the upper cell of the crozier by formation of two septa (Fig 2A; small arrow). These nuclei will fuse as the cell begins polarized growth to become an ascus (Fig 2A; long arrow). Wild-type fruiting bodies thus contain 100–200 asci within which meiosis and sporulation occur. When almost all asci from a perithecium contain four ascospores, there are almost no remaining croziers.

View larger version (83K): In this window In a new window Download PPT slide   Figure 2. Perithecium development in wild type and mutants. (a) Wild-type young perithecium with four-nucleated croziers (small arrow) and asci in prophase I (long arrow). (b) pah1 fpr1T66I croziers. All are uninucleate (arrowhead) except one, which shows four nuclei (arrow). (c) Two asci of fpr1T66I (R15).The ascus on the right shows a metaphase I with 7 chromosomes (large arrowhead) and the ascus on the left shows metaphase II nuclei with 14 chromosomes in total (small arrowhead). (d) fpr1T66I ascus with four ascospores. The middle ascospore contains two normal nuclei (large arrowhead). Nuclei from the two other ascospores are abnormal (smaller, arrowhead and condensed, arrow) due to aneuploidy. (e) pah1 fpr1T66I ascus with two ascospores each containing two nuclei (arrowhead points to the nucleolus). Bar, 5 µm.

smr2, fmr1, and fpr1 mutant fruiting bodies contain either no or only a few asci (1–50) and their development is abnormal in several respects.

1. Perithecia are always smaller than wild-type perithecia and a quarter of them contain only sterile paraphyses.

2. Another quarter, moreover, contain a few (2–30) uninucleate hook-shaped cells that are probably croziers, since some of their upper cells evolve into asci.

3. All other perithecia contain hundreds of "croziers," which almost never evolve into asci; instead, their nuclei divide and after septation their cells form long rows of more or less elongated cells that completely fill the perithecia. Moreover, in contrast to wild-type croziers (Fig 2A), almost all mutant croziers are uninucleate (Fig 2B). Half of those perithecia never differentiate asci.

4. When the upper cell of a uninucleate crozier forms an ascus, its nucleus enters meiosis. In those haploid meioses, the seven chromosomes are randomly distributed to the anaphase I poles, resulting in aberrant numbers of chromosomes in the four nuclei issued from the second division (Fig 2C). As some asci nonetheless proceed through postmeiotic mitosis (14 chromosomes are seen in such metaphases instead of the 4 x 7 expected in normal meiosis) and sporulation, the vast majority of ascospores are abnormal (Fig 2D). However, some asci contain four uninucleate or two binucleate (Fig 2E) wild-type-shaped ascospores that might result from early centromere cleavage at metaphase I giving rise to four nuclei with seven chromosomes.

A very small amount (<0.1%) of diploid croziers are also observed, either because septation does not occur after mitosis in a uninucleate crozier or because two nuclei are isolated within a crozier (see arrow in Fig 2B). These meioses are normal and give rise to four binucleate ascospores. Occasionally, the entire ascus yields a single giant spore.

Those three categories of perithecia are seen in all mutants. The number of perithecia and especially the number of large perithecia increase in the double mutants smr2 pah1, fmr1 pah1, and fpr1 pah1. However, their development is similar to what is observed with the single mutants. The phenotype of perithecia in crosses of fmr1-r pah1 or fmr1-r IncA with the wild-type mat- strain are similar to those of the self-fertile perithecia.

Phenotypes of strains deleted from a part of or from the entire fertilization domain:
The smr1-r fmr1^95-107 mutant strain (R10) carries a deletion of the FMR1 gene in the domain necessary for fertilization while the mat strain is deleted for the entire mat+ idiomorph (COPPIN et al. 1993 ). These two strains display a similar phenotype. They are unable to cross either with a mat- or a mat+ tester strain. Nevertheless, in a pah1 or in a IncA context, where the number of microconidia is enhanced, these two strains are able to act as the male partners for mat+ and mat- strains. The progeny are uniparental mat mutants, which indicates that only the mat mutant nucleus is able to enter the ascogenous hyphae and participate in ascospore formation (Table 6).

View this table: In this window In a new window   Table 6. fmr195–107 and mat crossed by wild type

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

FPR1, FMR1, and SMR2 are required for the repression and activation of functions involved in fertilization and internuclear recognition:
To date, neither wild-type strains of P. anserina nor any mat mutant has been found to be capable of fertilizing a strain of the same mating type. In contrast, fpr1 mutants described in this study are capable of selfing as well as of fertilizing a tester strain of the same mating type. Our interpretation is that these fpr1 mutant strains express the mat- genes required for fertilization. This implies that the wild-type FPR1 acts as a direct or indirect negative regulator of mat- fertilization genes (Fig 3). An equivalent phenotype has been observed with mat- strains with mutations in FMR1 and SMR2. The self-fertile phenotype of fmr1 and smr2 mutant strains is hardly detectable in a wild-type context but it becomes obvious when the microconidium number is increased, as, for instance, in pah1 strains (ARNAISE et al. 2001 ). This feature has delayed the detection of self-fertility in the initial examination of the fmr1-r and smr2-r mutants (ARNAISE et al. 1997 ) because the pah1 strain was not yet available. The self-fertile phenotype of fmr1 and smr2 mutant strains suggests that they have lost the ability to repress the mat+ fertilization genes and that the wild-type FMR1 and SMR2 are direct or indirect negative regulators of the mat+ genes required for fertilization (Fig 3). It is possible that the repressor activity of FMR1 and SMR2 on mat+ functions results from the formation of a FMR1/SMR2 heterodimer, as suggested by the interaction found between FMR1 and SMR2 employing the yeast two-hybrid system (E. COPPIN and R. DEBUCHY, unpublished results). In contrast to FMR1, SMR2 is not required for the expression of mat- mating type (DEBUCHY et al. 1993 ), but SMR2 appears to be a gene involved in fertilization in the sense that it is required during fertilization in mat- strains to avoid self-fertilization (Fig 3). SMR2 was not found to be expressed in the mycelium (COPPIN and DEBUCHY 2000 ), which suggests that this gene should be specifically expressed in mat- protoperithecia and microconidia, in addition to fruiting bodies where its transcription has been demonstrated (COPPIN and DEBUCHY 2000 ).

View larger version (12K): In this window In a new window Download PPT slide   Figure 3. Control of fertilization and IR in P. anserina. (A) In a mat- nucleus where only mat- specific functions are expressed. (B) In a mat+ nucleus where only mat+ specific functions are expressed.

In selfing perithecia of all fpr1, fmr1, and smr2 mutants, as well as in crosses between these mutants and a strain of the same mating type, fertilization is followed by the development of uninucleate croziers. Haploid meioses occur occasionally in these ascogenous hyphae and can lead to the formation of progeny consisting exclusively of the mat mutant nucleus. It is noticeable that the wild-type nucleus is never found in such progeny. Self-fertilization of fpr1, fmr1, or smr2 mutants suggests a straightforward explanation for this phenotype. We propose that the mutant nucleus expresses both nuclear identities, leading to self-recognition. Self-recognition would be sufficient to promote the developmental stages normally followed by two compatible nuclei. As for fertilization, wild-type FPR1 probably acts as a repressor of the mat- IR functions, as well as being an activator of mat+ IR functions (Fig 3). Conversely, FMR1 and SMR2 are probably repressors of the mat+ IR functions and activators of the mat- IR functions (Fig 3). Most of the fpr1, fmr1, and smr2 mutant genes remain competent to activate their specific IR functions but all of them have lost their repressor activity. Nuclear self-recognition may also explain the uniparental progeny that are produced in crosses of fpr1 and smr2 mutant strains to mat- and mat+ wild-type strains, respectively. This phenotype was reported initially by ZICKLER et al. 1995 for fmr1-1, smr2-1, and fpr1-1 strains in crosses to wild-type strains, but the authors did not propose any model for the formation of the uniparental progeny. We now can propose that the expression of the opposite nuclear identity in the mat mutant nucleus could trigger self-recognition, although the mutant nucleus remains competent for pairing with a compatible nucleus. If the mutant nucleus self-recognizes, it migrates alone into the ascogenous hyphae, ignoring its wild-type partner and eventually producing uniparental progeny. If the mutant nucleus pairs with a compatible nucleus, the pair follows the wild-type developmental pathway and yields biparental progeny. According to this model, the ratio of uniparental progeny mirrors the competition between self-recognition and pairing with a compatible nucleus. A high level of uniparental progeny could be attributed to a complete loss of repression of the alternative IR functions, resulting mostly in mutant nucleus self-recognition (e.g., the R12 mutant, Table 1). A low level of uniparental progeny could indicate that the repression of the alternative IR functions is mildly affected, which would favor pairing with a compatible nucleus instead of self-recognition (e.g., the R13 mutant, Table 1).

The hypothesis of the repression of alternative fertilization and IR functions by mat genes is supported by the phenotype of the mat and fmr1^95-107 strains. The mat strain carries a complete deletion of the mat+ information (COPPIN et al. 1993 ). The fmr1^95-107 mutant has a deletion within the domain that was previously characterized as necessary for the control of fertilization (DEBUCHY and COPPIN 1992 ). Both strains are nevertheless able to fertilize a mat+ or mat- wild-type strain, but the number of perithecia is 500–1000 times lower than the number of perithecia obtained in a wild-type cross, and these crosses yield no progeny (Table 6). The number of perithecia is increased and some ascospores are obtained when the number of microconidia is increased (in a pah1 or in an IncA context, Table 6). These observations indicate that the fertilization and IR mat+ and mat- target genes are expressed at a low level in a nonrepressed, nonactivated context. As self-fertility is not observed in wild-type strains, the mat+ and mat- functions should be utterly shut off in mat- and mat+ strains, respectively.

The type of smr1-r suppressor mutations and their implications in regard to fertilization and IR:
Mutageneses were performed during the vegetative state and expression of the smr-1 suppressors was necessary during the sexual cycle. This strategy requires that the suppressor mutations allow fertilization to start the sexual cycle. The excess of mutations in SMR2 is in agreement with the observation that this gene is not necessary for the expression of mat- fertilization functions. As expected, none of the three mutations in FPR1 alters fertilization. In contrast to the smr2 and fpr1 mutations, fmr1^95-107 has lost wild-type expression of the fertilization function and is supposed to have retained only the nonrepressed, nonactivated basal expression of these functions. Our experiments suggest that this basal expression is not sufficient to promote the sexual cycle unless fmr1^95-107 is associated with a mutation increasing the number of microconidia (Table 6). The most likely explanation for the presence of fmr1^95-107 among the suppressors is that it is a spontaneous mutation that occurred after fertilization instead of during mutagenesis.

How can nuclei expressing both nuclear identities bypass smr1-r developmental arrest?
It has been proposed that IR is followed by a developmental arrest (COPPIN and DEBUCHY 2000 ). Although this developmental arrest is not visible in a wild-type cross, it has been demonstrated by a lethal phenotype in ascospores that express the three IR genes. SMR1 allows these ascospores to recover from the lethal phenotype (COPPIN and DEBUCHY 2000 ). The developmental arrest is revealed in a smr1-r x mat+ cross by a block in the development of the perithecia (ARNAISE et al. 1997 ). According to this model, self-recognition of fpr1, fmr1, or smr2 mutant nuclei should result in a developmental arrest, while these nuclei can bypass the need for SMR1 and proceed through the developmental steps leading to a progeny. The reason for this by-pass is unknown. However, fmr1, smr2, or fpr1 mutants never give any biparental progeny in a cross in a smr1-r context, although these mutants are able to give at least some biparental asci in a cross in which a functional SMR1 is present in either parent. This result confirms that SMR1 is required for the recovery from the developmental arrest following IR between two compatible nuclei and it suggests that self-recognition might generate a mild form of developmental arrest that can be overcome without SMR1.

Comparisons with other fungi:
Little is known about the functions of mating-type genes in other filamentous ascomycetes. N. crassa has the same mating-type structure as P. anserina, but mutation analyses suggest that matA-2 (similar to SMR1) and matA-3 (similar to SMR2) have different functions from their counterparts in P. anserina (FERREIRA et al. 1998 ). In contrast, the expression of matA-1 and mata-1 in P. anserina, as well as the expression of FMR1 or FPR1 in N. crassa, suggest that fertilization is controlled in the same way in both fungi (ARNAISE et al. 1993 ). However, no self-mating or uniparental progeny have ever been reported in N. crassa as a result of mutations in mata-1 or matA-1 genes. It is possible that the basal expression of the target genes involved in fertilization and IR is so low that no repressor activity is required to obtain a complete extinction of the A and a target genes in a and A strains, respectively. The yeast S. cerevisiae offers a well-documented example that seems more related to P. anserina. The a mating functions are constitutive. In cells, the a-specific genes are repressed by MAT-2 while the -specific genes are induced by the MAT-1 gene product. This combination of repression and activation of target genes involved in mating is reminiscent of the events that occur during fertilization in the mat- strain of P. anserina (see Fig 3A), and the similarity of FMR1 with MAT-1 (DEBUCHY and COPPIN 1992 ) makes the similarity of the two systems even more striking. However, SMR2 and MAT-2 belong to different regulatory protein families; SMR2 is a HMG protein (DEBUCHY et al. 1993 ) and MAT-2 contains a homeodomain (SHEPHERD et al. 1984 ). Nevertheless, it is not known if SMR2 is a direct or an indirect repressor of mat+ fertilization functions and we cannot exclude that it activates the expression of a repressor similar to MAT-2. The mat+ mating system shows no similarity with the a mating system of yeast. In contrast to the a mating system, mat+ fertilization functions are not constitutively expressed, since their expression requires the presence of FPR1 (DEBUCHY and COPPIN 1992 ). However, the mat+ fertilization system operates in a similar way to the yeast mating system, except that FPR1 alone is sufficient to control the activation and repression of mat+ and mat- fertilization functions, respectively. We cannot exclude that, as for SMR2, the repressor effect of FPR1 is mediated by the activation of a repressor gene.

	ACKNOWLEDGMENTS

Marguerite Picard is gratefully acknowledged for her constant interest in this work and helpful discussions. We thank Evelyne Coppin for the gift of the mat^SK R1R5 strain.

Manuscript received February 8, 2001; Accepted for publication July 2, 2001.

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