Mol Gen Genet (1992) 233:113 121 © Springer-Verlag 1992

The mating types of Podospora anserina: functional analysis and sequence of the fertilization domains Robert Debuehy and Evelyne Coppin Institut de G~n~tiqueet de Microbiologie,U.R.A. D1354 du C.N.R.S., B~timent400, U.P.S. F-91405 Orsay C~dex, France Received October 3, 1991

Summary. The two idiomorphic alleles called mat + and m a t - , which control the mating types in Podospora anserina, have been cloned. M a t + and m a t - encompass 3.8 kb and 4.7 kb respectively, of unrelated DNA sequences flanked by common sequences. Subcloning allowed the identification and localization in each locus of the gene that controls fertilization, probably by determining the mating type. The mat + gene, called FPR1, encodes a protein with a potential DNA-binding H M G domain. The presence of this motif suggests that the FPR1 polypeptide may act as a transcriptional factor. The m a t gene called F M R 1 encodes a protein containing a motif that is also found in proteins controlling mating functions in Saccharomyces cerevisiae and Neurospora crassa. The role of this motif has not yet been established. Unlike the mat + locus, where the FPR1 gene seems to represent the major information, the m a t - locus contains information necessary for the post-fertilization steps of the sexual cycle besides the F M R 1 gene.

Key words: Fertilization - Fungus - H M G domain Mating types - Podospora anserina

Introduction Sexual reproduction of heterothallic filamentous fungi requires the interaction of two compatible sexual cells or organs. For a long time it has been known that this sexual compatibility is determined by gene(s) present at the mating-type locus, although the nature of these genes remained an enigma until the advent of cloning and sequencing techniques. At the moment, detailed investigation of the mating process is limited to Saccharomyces cerevisiae (for a review see Herskowitz 1989) and Schizosaccharomyces pornbe (for a review see Egel et al. 1990). In both yeasts the mating-type genes encode regulatory proteins, which in the case of S. cerevisiae, have Correspondence to. R. Debuchy

been demonstrated to influence the transcription of target genes (Bender and Sprague 1987). However no common scheme is applicable to the S. cerevisiae and S. pombe mating systems, and new regulatory pathways for the control of mating and sporulation may be expected in filamentous fungi. Moreover, filamentous fungi differentiate sexual organs which may add another layer of complexity to the expression of mating-type genes. We have initiated a study of the control of sexual reproduction in the filamentous ascomycete Podospora anserina by cloning the two alternative alleles, mat + and m a t - , which determine sexual compatibility. A haploid strain of P. anserina differentiates both female organs and male gametes but cannot self-fertilize. Fertilization occurs only between sexual structures of opposite mating types and initiates a series of differentiation events resulting in the formation of fruiting bodies which contain hundreds of ascospores. The interval between caryogamy and meiosis differs in yeasts and in P. anserina or other closely related fungi (e.g. Neurospora crassa). While in yeasts mating is immediately followed by caryogamy, in filamentous Ascomycetes this event is preceded by a series of mitoses that occur first in syncytial structures, then in dikaryotic hyphae, where the male and female nuclei finally fuse. Conversely meiosis can be postponed after caryogamy in yeasts, leading to vegetative diploid cells, whereas in P. anserina diploid nuclei immediately engage in meiosis followed by the formation of ascospores. The numerous premeiotic divisions occurring after fertilization allow multiplication of the number of asci that originate from one fertilization event. Molecular analysis of the mating-type genes is the first step in determining the regulation of these complex events. A structural analysis of the mat genomic region of P. anserina is presented in this paper, in which we show that mat + and m a t - correspond to non-homologous sequences present at a homologous site on the chromosome. The same situation was previously reported for the A and a alleles of N. crassa (Glass et al. 1988). The term 'idiomorphs' was proposed by Metzenberg and Glass (1990) instead of 'alleles', which seems inappropriate to these

114 structures. The functional analysis of m a t + and m a t idiomorphs has allowed the identification in each m a t locus of putative genes called F P R 1 (for Fertilization Plus Regulator) and F M R 1 (for Fertilization Minus Regulator) respectively. These genes have been demonstrated to be necessary for fertilization, probably because they determine the mating type of sexual cells. The F P R 1 gene encodes a polypeptide with a potential DNA-binding H M G domain (see Jantzen et al. 1990), suggesting that this protein may be a transcriptional factor involved in the control of mating type. The deduced FMR1 protein shows similarities with proteins controlling mating type in N. crassa and S. cerevisiae. Sequences necessary for steps subsequent to fertilization were also identified and localized to the m a t - locus. Materials and methods P. anserina strains and culture media. The solid and liquid growth media for P. anserina were described by Esser (1974). A m a t - S strain was used for the isolation of the m a t - locus and a m a t + s strain was used for the isolation of the m a t + locus. The recipient S strain for transformation experiments carried the l e u l - i U G A mutation, which is suppressed by the opal suppressor tRNA su8-I (Debuchy and Brygoo 1985). Plasmids and bacterial strains. Escherichia coli strain

HB101 (Boyer and Roulland-Dussoix 1969) was used as the recipient strain for all recombinant plasmids containing m a t - DNA. Subcloning of this D N A for functional analyses was done in plasmid pHSU8 (Debuchy et al. 1988). The plasmid pHSU8 has the P. anserina su8-1 suppressor, which encodes a serine tRNA reading the U G A stop codon (Debuchy and Brygoo 1985), functional ampicillin and chloramphenicol resistance genes and a cos site. Cosmids N10 and N9 are pHSU8 with m a t inserts in the E c o R I site in the chloramphenicol resistance gene. E. coli strain CM5a (Camonis et al. 1990) was used as the recipient strain for all recombinant plasmids containing m a t + DNA. Subcloning of this D N A was performed in pUC18 (Yanisch-Perron et al. 1985) or Bluescript (Stratagene) cloning vectors. Plasmid pM33-18M3 contains the leul + gene of P. anserina cloned by Turcq (1989) in pUC8. P. anserina transformation. Transformation assays were performed as previously described (Picard et al. 1991). The m a t - genomic region was subcloned into pHSU8, allowing direct transformation of the leul~l m a t + recipient strain. Ten to twenty Leu + transformants obtained with each construct were subjected to mating tests to determine the three phenotypes: m a t +, m a t - and m a t + m a t - (namely, self-mating). We concluded that a recombinant plasmid contained functional mating information when most transformants were dual maters and selfmaters. Self-mating corresponded to the differentiation of fruiting bodies on the haploid thallus of the transformed strain regardless of the production of ascospores which, in fact occurred very rarely, if at all. This self-

mating prevents the use of the transformants as female parents in a cross. The function of cloned m a t - information was therefore investigated by crossing the transformants as male parent with a m a t + tester in order to determine whether mature fruiting bodies could be obtained. Some transformants only produced small fruiting bodies that were devoid of ascospores, whereas others were able to produce mature fruiting bodies with progeny. Although these last transformants could be considered as fertile in crosses with a m a t + tester strain, only a minority of fruiting bodies produced ascospores and the progeny were not as abundant as in a conventional cross. In contrast, when the resident m a t + information was involved in the cross (transformant as a male x m a t - as a female), normal progeny were obtained. Dissymetry in the expression of resident m a t information and added information has already been reported (Picard et al. 1991). The m a t + region was first subcloned into pUC18 and subsequent subcloning was carried out using Bluescript phagemid. These vectors do not contain a marker allowing selection in P. anserina; thus they were cotransformed into the l e u l - l recipient in association with the pM33-18M3 plasmid containing the leul ÷ gene of P. anserina (Turcq 1989). In each transformation assay about 100 Leu + transformants were analysed as described above. When the recombinant vector contained functional m a t + information, transformants with the dual- and self-mater phenotypes were recovered at a frequency of 10 to 20%, corresponding to the usual cotransformation efficiency. These transformants were then tested in crosses with a m a t - tester as explained above. Nucleotide sequence determination. The H i n d I I I - B a m H I and the B a m H I - P s t I fragments of the m a t - idiomorph

were isolated from the cosmid N l l (Picard et al. 1991) and introduced into Bluescript KSM 13 + and Bluescript SKM13+ (Stratagene). The S a l I - E c o R V fragment of the m a t + idiomorph was cloned into Bluescript S K M 1 3 + . The recombinant vectors were used to produce an ordered set of deletion clones according to the method described by Henikoff (1984). The pBluescript SKHH containing the H i n d I I I fragment of m a t + (Fig. 2) allowed determination of the D N A sequence of the 320 nucleotides between the H i n d I I I and SalI sites. The D N A sequence was determined for one strand by the dideoxynucleotide chain termination method (Sanger et al. 1977) with T7 D N A polymerase (Tabor and Richardson 1987). The sequence of the complementary strand was obtained using oligonucleotides synthesized by the phosphoramidite technique with an Applied Biosystems 381A D N A synthesizer and the method mentioned above. Results Isolation o f functional mat + and mat- f r a g m e n t s f r o m within mat-specific sequences

Cosmids covering the m a t - locus were recently isolated by hybridization with the m t A - 1 gene of N. erassa (Pi-

115

card et al. 1991): all the selected cosmid inserts had an 18 kb E c o R I fragment which hybridized with the probe. Further genetic and physiological analyses demonstrated that this fragment contained the information necessary for fertilization. The 18 kb E c o R I m a t - fragment hybridized with a 22 kb E c o R I m a t + fragment, which is present at the m a t + locus. This 22 kb E c o R I fragment turned out to have the information necessary for mating and gave no hybridization signal with the m t A - 1 gene. The data suggested that the m a t - locus contained a sequence specific for m a t - , which could be cloned by differential hybridization. A S a u 3 A library of a m a t cosmid was hybridized successively with the 18 kb m a t and 22 kb m a t + fragments and a recombinant plasmid hybridizing only with the m a t - probe was isolated. A plasmid with a mat+-specific sequence was isolated after screening a S a u 3 A library of the m a t + cosmid with the same two probes. This confirmed that the two m a t loci contained unrelated sequences. Larger fragments were isolated according to the following strategy. The m a t and m a t ÷ cosmids were cut with different restriction enzymes before transformation, to determine which of them inactivate the gene conferring mating specificity. Further identification of the functional fragment among the complex digestion products of the cosmids was facilitated by hybridization with the Sau3A-specific fragments. This rationale allowed us to clone a 1.9 kb H i n d l I I - B a m H I fragment and a 2.2 kb S a l I - E c o R V fragment which, upon transformation, confer a Matand Mat + mating phenotype respectively (see below). Their localization on the restriction map of the m a t locus is presented in Fig. 1 and 2.

marker and the specific m a t - specific 1.9 kb H i n d l I I B a m H I fragment. Most of the m a t + transformed strains were found to have acquired the m a t - fertilization ability, indicating that the information for this function is included within the H i n d I I I - B a m H I fragment. Although fruiting bodies developed when these transformants were crossed with a m a t + tester, mature ascospores were never produced. This phenotype indicates that the H i n d I I I B a m H I fragment does not contain all the information necessary for the completion of the sexual cycle. Previous experiments (Picard et al. 1991) had demonstrated that this additional information is present in the N9 cosmid and absent from the N10 cosmid. Therefore, other plasmids were constructed to extend the subcloned fragment on both sides (Fig. 1) and tested for the presence of information sufficient for complete sexual reproduction by transformation. The mating behaviour of the transformants is reported in Fig. 1. Analysis of transformants obtained with pHMTEB indicates that extending the subcloned fragment towards the left E c o R I site does not allow completion of the sexual cycle. Only strains transformed with the 5.7 kb P s t I - P s t I fragment which overlaps the initial H i n d I I I - B a m H I fragment were found to give progeny with a m a t + partner. This indicated that no information necessary for the sexual cycle is present in the homologous part of the m a t - locus, beyond the leftmost P s t I site. It is concluded that the m a t - idiomorph can be divided into three functional domains: the H i n d I I I - B a m H I fragment contains information necessary for fertilization and domains on each side of this fragment have information necessary for the steps following fertilization.

Functional analysis o f the mat- locus

Functional analysis o f the mat + locus

A l e u l - 1 m a t + recipient was transformed with the plasmid p H M T B H (Fig. 1), which carried the su8-1 selective

A l e u l - 1 m a t - recipient was cotransformed with the Ieul + gene and the vector KSSRV containing the S a i l -

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Fig. 1A-C. Functional analysis of the m a t - locus. A Restriction map o f chromosome I at the m a t - locus, the thin line represents sequences common to m a t - and m a t + strains, the t h i c k line represents m a t - specific sequences. The centromere-proximal end is at the left of the map. B D N A fragments subcloned in plasmid pHSU8. Each b a r corresponds to a restriction fragment o f the above map. N9 and N 10 are cosmid inserts which extend to the right side of the map (Picard et al. 1991). The recombinant plasmids were introduced into a m a t + leul-1 strain and Leu + transformants were

tested for the ability to fertilize a m a t + tester strain (indicated by: + for fertilization and - for no fertilization under the letter F) and for the ability to give ascospores on a m a t + tester strain (indicated by: + for ascospore formation and - for no ascospores under the letter A). C The b a r s indicate the position of the relevant information as deduced from subcloning and transformation experiments. The a r r o w indicates the position of the F M R 1 gene as deduced from the sequences data

116 Dra]]

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Fig. 2A-C. Functional analysis of the m a t + locus. A restriction map of chromosome I at the m a t + locus: the thin line represents sequences c o m m o n to m a t - and m a t + strains, the t h i c k line represents m a t + specific sequences. The centromere-proximal end is at the left of the map. B D N A fragments subcloned in pUC18 (puc EP) or in Bluescript (KS SRV, SK SS, SK R I R V and SK HH). The recombinant plasmids were co-introduced into a m a t l e u l - 1 strain with

a plasmid containing the l e u l + gene and Leu + transformants were tested for the ability to fertilize a m a t - tester strain and for the ability to give progeny on a m a t tester strain. Symbols are as for Fig. 1B; _+ under the letter A indicates low production of ascospores. C As for Fig. IC except that the a r r o w represents the F P R 1

E c o R V fragment of the mat + locus (Fig. 2). Leu + transformants with the ability to fertilize a m a t - tester strain were recovered. Since most of the fertilized m a t - strains

the corresponding gene of P. anserina; such similarities were found in the 5' coding region of these two genes and confirmed the existence and position of the first intron, which interrupts the coding sequence of F M R 1 and mtA-1 in exactly the same nucleotide context. The lengths of the introns in the F M R 1 and mtA-1 genes are 60 and 58 bp respectively with no significant sequence similarity. Another putative intron was localized in the 3' end of the coding sequence of F M R 1 but cannot be confirmed by comparison with mtA-1 because these genes are not similar in this region. Alignment of the 5' non-translated regions of F M R 1 and ratA-1 identified the conserved box CAACTTCG, which is located 87 bp and 81 bp 5' to the ATG of F M R 1 and m t A - 1 0 R F s , respectively. Comparison of the deduced polypeptides of the F M R 1 and mtA-1 genes shows that out of the 196 N-terminal amino acids, 106 are identical (Fig. 4A), whereas the C-terminal parts of the proteins are dissimilar. A sub-region of 53 amino acids common to the F M R 1 and mtA-1 proteins also displays similarities with the al protein of S. cerevisiae (Astell et al. 1981; Tatchell et al. 1981) (Fig. 5). We propose to call this motif the a-domain. A similarity search carried out with the FASTA program

were sterile or poorly fertile, fragments larger than the S a I I - E c o R V fragment were tested for an increase of ascospore production. Transformation using a larger fragment that extends on the SalI side up to the HindIII site was necessary and sufficient to allow m a t - recipients to mate and give ascospores upon crosses with m a t strains with the same efficiency as transformants containing the complete idiomorph (Fig. 2).

The region specific f o r mat- contains a gene necessary for fertilization and displays similarities with already known mating-type regulatory proteins

The nucleotide sequence of the H i n d I I I - B a m H I fragment containing the m a t - fertilization information was determined on both strands. Analysis of this sequence allowed the identification of a putative gene called F M R 1 (Fig. 3A). As the H i n d I I I - B a m H I fragment contained only the 5' part of this gene truncated at the B a m H I site, sequencing was performed on both strands through the B a m H I site toward the PstI site. The coding sequence of F M R 1 may begin at a methionine codon surrounded by a perfect translation start consensus (Ballance 1986). A possible intron was identified by consensus sequences for the 5' and 3' splice sites (Ballance 1986) downstream from the the translation start. The F M R 1 sequence was compared with the sequence of the mtA-1 gene which controls the A mating type in N. crassa (Glass et al. 1990a). This latter gene is a part of the A idiomorph and was used as a probe for cloning the mat allele of P. anserina (Picard et al. 1991), and therefore a high degree of sequence similarity was expected between ratA-1 and

gene

Fig. 3 A and B. Sequences of the m a t specific genes controlling fertilization in P o d o s p o r a anserina. A Sequence of the F M R 1 gene of the m a t - idiomorph. The nucleotide sequence is numbered from the centromere proximal end of the m a t - idiomorph. The amino acid sequence of the F M R 1 gene is shown below the nucleotide sequence. The putative introns are b o x e d : F M R 1 and F P R 1 comm o n boxes in the 5' non-translated regions are underlined. B Sequence of the F P R 1 gene of the m a t + idiomorph. Legend as for Fig. 3A, except that this sequence is numbered from the D r a I I site towards the centromere-proximal end of the idiomorph

117 A

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AGGCGAAACTGATGTCGGCTCACC•TCATTATCGCTATGTGCCTCGTCGATCTTCTGAGATC•GCCGCCGCGCTCCCCGC•GTAACCGAG K A K L M S A H P H Y R Y V P R R S S E I R R R A P R R N R

1171

CACAGGAAGTCGCCAATGCTTC•CCGATCGGGGAGAACTCGGGTGCCCCTATCGTAGGCAATCCTATTGTCA•CAC•ATGGAGCAACAGC

1~I

AGC•CCTTCCTGACAT•AGTATTGCCCCTAACCAGGAGATCACCAAGGACAACGATGTCAGCCATCTCATCGACCCTCCCCATGTCTTCT Q P L P D I S I A P N Q E I T K D N D V S H L I D P P H V F

1351

CTGGTCAGATTACTGAGCTCATGCCCGATGTGGCGAACTT•CTGCCTCCTATGATACGCGAAGGCTGGTCTCCTCTT•ATGACTTCCG•G S G Q I T E L M P D V A N F L P P M I R E G W S P L H D F R

1441

CTGTTCTGAATGGA•A•A•TGGAAACAATGGAGTCGACTGTG•TCTTACTCCAGAGTCTGAATCCCAGGATGACTTTGTCGGTACT•C•T A V L N G H T G N N G V D C A L T P E S E S Q D D F V G T P

1531

CCTCCACGATGCCTGA•AACAGTGCCTTCGATTGGATCACTGGAACGGAGGAGGATTTGGCCCAAAT•TTCGGTCAATTCTGATCACTTC

1621

CG•GGC•TCTTTCGGAA•AC•AGCTCTTGACA•TATT•AGT•ACTG•GACCTTTCGCA•AAGGTTTG•CGCCCAATGGGCGATGACCTAT

1711

GCAC•AGACCTTGGTT•AGAATGGTAAGTCGTATCACCTCTCTCTCTCTCTACA•ACGGTTGCTGACCTCT•TTCTAGCTTCAAAGCTT

O

A

S

A

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A

N

D

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G

A

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Hi n dill

118 A FMR1 M A[G]~:~:~]:NS[~ ~:.Q [ ~ E G [ ~ [ ~ G [ ~ AET L[~ D [ ~ - ~ E[~-T] P R Q mtA-1 M SIGINiD Q[I[N KIT FIA DIL[~[EIDID RIE A A MI!:~i~IFISIRIMM RIRIG TIE P V - -[A[ K K K V N G FFM O [ i ~ [ ~ ' ~ - - ~ S M[FS Q L FMR1 R P R i P A A K K K V N M G [..... ~[RSYYS[PL[FsQLPQ mtA-I

K ERS [ C[~ Q K E R S P FPI[ MIT]

FMRI T ~ Q [ ~ - ~ - ~ K ~ A [ ~ A ~ D Q{~A E mt A-1 II L W QIHID P F HIN]E W D F M C] SIV W S, S[I RIT YILIE Q Q EN

~

T~-~]

cause m a t + transformants to fertilize m a t + tester strains (data not shown). These results confirm that this gene is necessary for fertilization and enable localization of the domain ensuring this control within the first 229 residues of the 305 or 349 amino acids of the total F M R 1 protein.

T['~ Q[R]M D FMRI Qi:~[X~]T P [ ~ i ~ : ! ~ : PI [ ~ DT GN [ ~ A E A S C [ ' ~ V[~ W~] L D D [ ~ rata-1 H i ~ G HIL GI~::[~ FIGVvlN[LIVIRIF P NIG T HIDIHEIRIT A FMR1 i~[~ D~:~:~:IR ~ Y H L Q P M N G L O[~ F L S [ ~ N O ~ ! i i F D~] Q N [ i [ ~ S Q['~ rnt A-1 il)l.PJL!~ Q n NIL Q P MN O L CILIL WKIC LIE SIO L P[~IiA NIPIH S ~ I I I A KIL FMR1 S D P Ai~[~¥[~]C I['~T V P ~ I P [ ~ T F D TM S G F R Qi~[~iK Q N P A L mt A-1 S D P l SI~IDIM[ IIW FIN[K Q P H::R:.Q QIGIH A V Q T D E S E Vi~::!~iiS A M F P R

B FPR1 M A A F N F E A F

S LTPQGS

FPR1 O S F O Y O N R A F Q F D F A S mt a-1

T I SAAPRPAAPA

I DRTVQQQC

L E S L P E D A N P G ~ : T E ~ L [ ~ A K Y ~ -] M D O N S T H P ~ P N ~ KLTAT M AIW[

FPRI N H F~] I [ ~ - - ~ f f ~ T L [ ~ " ~ } ~ : ~ D:~IQM[F] S I M P D H T K K['~.~:~N T rata-1 S R IISIN]Q L O HWN[D R[K V l[N!}i P~::S DIFIL N T H P D I Q S[Gli~i ::I:::iAE FPR1 M N T T V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mta-1 F K K A T G E E G M F A R D P E S LG IMLLG PVKL FKPDS VVVD FPR1 . . . . [ W ~ - ~ K R[~eiQ['~V A [ ~ - -[AKI P R P PNAYI rata-1 G N L F [ W D P K [ O IIH[~!iS[AIPKIEIQQKKAK I P R P P N A Y I

LYRKDJ LYRKD

FPR1 Q Q R A A ~ i A A[-N-'-~ ~i; P['N~ 0 [ ~ - ~ M T { ~ G [ M ~ K~] S{~ E i~['R]A E ~ Q rata-1 H H Ei~:~ :' E Q[N P G[~]H[N N[~[ I S "7[ I V[G]N[MVvl~ DIEIQIPIHi~[R[E K F FPR1 R R A S[E-~A~!i~[~M S A H [ - ~ H ~ - ' Y ] V ~ S E [ ' f - ~ ] i ~ i V R R N:iRI!~ rat a-1 NM S NIE I KIT ~ILIL L E NIHDIY R YINIP R R SlQ D[ I R R RJ~{iS P Y L!!~ i~!i:

Fig. 4A and B. Comparison of the deduced polypeptides of the genes controlling fertilization in P. anserina and Neurospora crassa. Only similar regions are shown. A Alignment of the polypeptides deduced from FMR1 and ratA-1 (Glass et al. 1990a). In some regions the alignment requires gaps which are indicated by dashes. Identical amino acids are boxed; conservative changes of amino acids are shaded. Amino acid changes are considered as conservative when they follow the chemical alphabet (Mahler and Cordes 1966). B Alignment of the polypeptides deduced from FPR1 and mt a-1 (Staben and Yanofsky 1990). Legend as for Fig. 4A (Pearson and Lipman 1988) in the Swiss-Prot data bank Version 18 did not reveal other conserved region in FMR1.

Several arguments indicate that the control of fertilization ability can be attributed to the F M R 1 gene. During subcloning, some restriction enzymes, such as ClaI or E c o R V , have been found to inactivate the fertilization information. Sites for these enzymes were localized close to or within the coding sequence of F M R 1 (Fig. 3). However, curing at some restriction sites inside the F M R 1 gene does not cause inactivation, as evidenced by the H i n d I I I - B a m H I fragment which has active fertilization information (Fig. 1) although the B a m H I site is localized inside the F M R 1 coding sequence (Fig. 3). This H i n d I I I - B a m H I fragment, which contains the 5' end of the F M R 1 coding sequence, was subjected to nested deletions starting at the B a m H I site to determine whether deletions inside the remaining F M R 1 coding sequence can inactivate the fertilization function. A derivative fragment, which lacks 215 nucleotides at the B a m H I side within the F M R 1 coding sequence, cannot

The region specific f o r mat + contains a gene essential f o r fertilization, which has similarity to the H M G class o f D N A - b i n d i n 9 proteins

The nucleotide sequence was determined on both strands of the E c o R V - H i n d I I I fragment conferring mat + fertilization ability. Analysis of the sequence revealed three ORFs (Fig. 3B) exhibiting a high degree of similarity with the m t a-1 polypeptide of N. crassa (Staben and Yanofsky 1990). The ORFs might correspond to a unique coding sequence, which is interrupted by two introns of 163 and 53 nucleotides, the ends of which were determined by matching with splice consensus sequences. The position of the second intron is strongly supported by analogy with N. crassa: it interrupts the O R F at the same nucleotidic position in the F P R 1 gene of P. anserina and in m t a-1. The coding sequence may start at a methionine codon at position 300 which has the correct context of a translation initiation codon. The following arguments suggest that the proposed gene, called F P R 1 , corresponds to the information conferring the m a t + fertilization function. (i) A plasmid carrying the E c o R I E c o R V fragment was found to maintain its m a t + information after transformation, when digested with DraII, which has a unique site located 300 bp upstream of the putative translation start. (ii) The plasmid lost the m a t + information when digested with A v a I I or B a m H I , which cut in the first and in the second exons respectively. These results agree with the positions of the respective restriction sites outside and inside the putative F P R I gene. (iii) The m a t + fertilization information is lost if the S a l I - E c o R V fragment has a deletion of 600 nucleotides at the SalI site. This deletion is inside the coding sequence of the proposed F P R 1 gene. Although the coding sequence of the F P R 1 gene continues beyond the SalI site, the S a I I - E c o R V fragment is sufficient for fertilization, indicating that the carboxylterminus o f F P R 1 is dispensable for m a t + fertilization function. Extension of m a t + S a l I - E c o R V fragment beyond the SalI site is necessary to recover transformants of a m a t - recipient that are able to produce a significant number of ascospores when crossed to a m a t - strain (Fig. 2). This suggests that the carboxyl-terminus of the F P R 1 protein is necessary to confer fully functional m a t + behaviour, but the evidence is presently inadequate to

m~*-~ 44 IK KIKii~INpV MI~::::::~IRISlY V SIP L FIs[QI:~:=PIqK E RIS V r M • ~ L1','1¢ HtDV F H N E -IW@iM C S VlY I S SI~ 1 96 c~l

90 [K K]¥ ~]NIS FL~.~i~{~AIY Y SIQ F OIS~G.i~.KIQJNV LISIS L L A E E[.~I-i A[DIK M Q H O I]W D]~!:F A Q Q[YIN F~I] 143

Fig. 5. Alignment of the amino acid sequence defining the a domain of proteins encoded by the following genes : FMR1 of P. anserina, mtA-1 of N. crassa (Glass et al. 1990a) and MATCH of Saech-

aromyces cerevisiae (Astell et al. 1981; Tatchell et al. 1981). The

numbering indicates the position of the first and last amino acids of the peptide within the protein sequence. Legend as for Fig. 4A

119

FPR1 133AKI P~PP NRYI Lr~R K~QQAA L KAAN~G I P NNDRS V T i ~ G q K K . . . . 1 I14 AK I PIRIP P N]qY I L]YIR KID[HHR E

E S P E VRA Er~ 184

I REQNIPpLHNNEIIIS v I vlo[N~VJaD E Q P H ~1~1~~R ~65

~c ~01~PI,I~PqqFILIYI~KRK~*~LL~N~ ~N~;~OlVISK~VlOI~I~,~K~VI~IMqYII~

SRY Dr~vKIRIPMNIqFI VMSR[qQRRKMALENIPIRMRNS q qS KQLIGIYQIVIKMLWEAEIKIWPIFI HMG1 93 N A P K ] R ] P P S I A I F F L ~ F ] C S I E ] Y R P K I K G E H ] P ] O L S IGDIVIAKKLICIE~V~NNTAADDNHPIYI144 hUBF 405EKPK[R]PVS]A]MF I ] F ] S E I E ] K R R Q L Q E E R ] P ] E L S E S E ] L ~ T R L L I A ~ R M ~ N D L S E K K [ K ] A K ] Y ] 4 5 6

Fig. 6. Alignment of amino acid sequence defining the H M O domain of proteins encoded by FPR1 of P. anserina, mt a-1 of N. crassa (Glass et al. 1990a), M A T M c from Schizosaccharomyces pornbe (Kelly et al. 1988), human S R Y(Sinclair et al. 1990), the gene for the pig H M G 1 (Tsuda et al. 1988) and human nucleolar tran-

scription factor, hUBF (Jantzen et al. 1990). The human S R Y encoded peptide has no numbering because the complete protein sequence is still unknown. The amino acids that belong to the H M G domain consensus (Sinclair et al. 1990) are boxed

justify a definite conclusion. Another possible candidate gene for the property attributed to the end of the F P R 1 protein is represented by an O R F of 713 bp present in the non-coding strand of the F P R 1 gene. This ORF is localized in the region corresponding to the 3' part of F P R 1 and is totally included within the DNA segment containing F P R 1 . Since this O R F cannot be physically separated from the F P R 1 gene, further experiments are necessary to determine if this long ORF corresponds to a gene controlling a step in the sexual cycle. Interestingly, a protein-coding region of 621 bp is present in the noncoding strand of the end of the m t a-1 gene of N. crassa, but this O R F has no similarity with the ORF present in the non-coding strand of F P R 1 . As mentioned above, identification of the F P R 1 polypeptide was facilitated by its extensive similarities with the m t a - 1 0 R F of N. crassa (Fig. 4B). Similarities were found in the central part of the 365 amino-acid sequence of F P R 1 , and include a motif called the H M G domain which was first characterized in the human upstream binding factor of ribosomal R N A genes (Jantzen et al. 1990). The H M G domain is present in a set of proteins suggested or known to be regulatory proteins, for instance the protein encoded by the sex determining region of chromosome Y in mammals (Sinclair et al. 1990; Gubbay et al. 1990) or the M A T - M c protein of S. p o m b e (Kelly et al. 1988; Fig. 6). A search in the Swiss-Prot bank Version 18 with the FASTA program (Pearson and Lipman 1988) did not identify any other conserved region in protein F P R 1 . The sequence upstream to the ATG of F P R 1 and F M R 1 were compared for common sequence which could be the target of putative common regulatory factors. The sequence CAACTTC, which is located 34 bp 5' is also present 82 bp 5' to the to the ATG o f F P R 1 0 R F ATG of F M R 1 0 R F . The same sequence is found 81 bp 5' to the ATG on ratA-1 (Glass et al. 1990a). A search for other boxes conserved in the ATG upstream sequence of F P R 1 and F M R 1 allows the identification of the sequence GTCTTTCT which is 222 bp and 199 bp 5' to the ATG of F P R 1 and F M R 1 , respectively. It is not yet known if these boxes are important for the transcriptional or translational control of F P R 1 and F M R 1 expression.

(Picard et al. 1991). Analysis was pursued by subcloning experiments followed by transformation assays, which established the localization of the sequence involved in the fertilization functions in the cloned DNA. Hybridization analyses indicated that m a t + and m a t - loci contained dissimilar D N A sequences and preliminary sequencing data (not show) allowed us to delimit the boundaries of the specific m a t + and m a t - sequences and to determine their size. The m a t - and the m a t + idiomorphs are 4.7 kb and 3.8 kb long respectively. Dissimilar sequences in a homologous locus are also a structural feature of the mating-type locus of S. cerevisiae (Astell et al. 1981; Tatchell et al. 1981), S. p o m b e (Kelly et al. 1988), N. crassa (Glass et al. 1990a; Staben and Yanofsky 1990) and of the Basidiomycetes SchizophylIum c o m m u n e (Giasson et al. 1989) and Coprinus cinereus (Mutasa et al. 1990). In fact each haploid cell of S. cerevisiae or S. p o m b e has a silent copy of each idiomorph in addition to the idiomorph present in the expressed locus (reviewed in Herskowitz 1989 and Egel et al. 1990 respectively). This raises the possibility that the species with a unique idiomorph per haploid genome have evolved from organisms with a copy structure, through the loss of the silent copies. The functional analysis of the m a t - specific region led us to isolate a fragment which confers on a m a t + recipient strain the capacity to fertilize itself and a m a t + tester strain. Transformants containing this fragment acted as m a t - male parents in a cross but their capacity to differentiate female organs with the m a t - specificity could not be determined because of self-fertilization. Fertilization function is thus restricted to the male function in the following analysis. A fragment containing the m a t + fertilization was obtained by the same method. Putative genes, called F M R 1 and F P R 1 , were characterized in the m a t + and m a t - fragments respectively. These genes were demonstrated to be necessary for fertilization. The F M R 1 gene has a region of similarity to the M A T c ¢ I polypeptide ofS. cerevisiae and to the m t A - 1 polypeptide ofN. crassa. Neither the whole F M R 1 nor the complete ratA-1 gene is required for fertilization. In each case the 228 N-terminal amino acids are sufficient to confer mating control, but the F M R 1 and m t A - 1 proteins lost this function if they were reduced to 158 and 184 amino acids respectively. These data suggest that the region of similarity between F M R I and m t A ~ l polypeptides might be involved in a common function, namely the control of fertilization, but the mechanism of this control remains unknown. The F P R ! gene, assumed to be responsible for the m a t + fertilization function, en-

Discussion

Previous work allowed us to clone the genomic regions of P. anserina containing the m a t + and the m a t - loci

120

codes a polypeptide that displays similarities with the m t a-1 polypeptide of N. crassa. The similarities are localized in the amino-terminal part of the m t a-1 polypeptide which was proposed to control fertilization in N. crassa (Staben and Yanofsky 1990). Some of the amino acids common to the two proteins belong to a motif called the H M G domain, which was found initially in the human nucleolar transcription factor h U B F (Jantzen et al. 1990). The presence of the same motif in the F P R 1 protein suggests that it may be a transcriptional regulatory protein. Alignment of the F P R 1 and m t a-1 genes within the region of the 470 bp encoding similar polypeptides indicates that 56% of the nucleotides are conserved. The same alignment was also done with approximately 600 bp encoding the similar regions of the F M R 1 and m t A - 1 polypeptides and the analysis indicates that 62% of the nucleotides are conserved. Although the numerical difference is low, hybridization of F P R 1 and m t a-1 DNAs produces a very weak signal (data not shown), whereas hybridization of the DNAs from the other pair gives a clear signal (Picard et al. 1991). This situation can be related to the situation found in the genus Neurospora in which homothallic species were tested for the presence of a sequence similar to the A and a idiomorphs of N. crassa (Glass et al. 1988, 1990b). Most of them contain sequences that hybridize with the A-specific probe, whereas the a-specific probe failed to give any signal. The results with P. anserina raise the possibility that these strains contain an idiomorph that is genetically equivalent to a although not detectable by hybridization. Comparison ot the m a t genes from P. anserina and N. crassa gives some indication of their evolutionary relationship. The position of the first intron in the F M R 1 and m t A - 1 genes is perfectly conserved, suggesting that these two genes diverged from a common ancestor. The same conclusion was suggested by comparison of the F P R 1 and m t a-1 genes which have conserved the position of the second intron. An analysis of the entire idiomorphs of P. anserina and N. crassa will be necessary to establish whether these sequences have a similar overall structure and diverged from a common structure. A first step towards this analysis was accomplished by determining orientation of the idiomorph relative to the centromere in P. anserina (S. Arnaise and A. Adoutte, personal communication) and in N. crassa (Glass et al. 1990a; Staben and Yanofsky 1990). The F M R 1 gene and its counterpart in N. crassa are both localized at the centromere-distal side of the region of heterology and both are oriented toward the telomere. The F P R 1 and m t a-1 genes are localized on the centromere-proximal end of the heterologous region and are oriented toward the centromere. If the idiomorphs have a similar structure determining a heterothallic sexuality in P. anserina and N. crassa, it is likely that these fungi have evolved from a heterothallic ancestor. This would imply that the homothallic species found in Podospora (Rizet 1940) and Neurospora (Glass et al. 1990b) arise from heterothallic organisms. While the idiomorphs in P. anserina and N. crassa contain several kilobases of DNA, only a part of these

specific sequences is required for determining mating identity in the fertilization process. Sequences upstream and downstream of the minimal F M R 1 region conferring the Mat- fertilization phenotype are necessary for the development of mature fruiting bodies in crosses with m a t + strains. Preliminary results suggest that at least one gene controlling post-fertilization events may be present in the upstream region of F M R 1 . The other fragment downstream from the the fertilization domain of F M R 1 contains the 3' coding region of F M R 1 . Experiments are in progress to test the possibility that this 3' part of F M R 1 , which is not necessary for fertilization, could be involved in the control of subsequent steps. The situation of the m a t + idiomorph is different. While the m a t - idiomorph contains distinct information controlling fertilization and subsequent events, the F P R 1 gene seems to be the major m a t + information necessary to obtain a fertile m a t + strain. The dissimilar structure of the idiomorphs for the control of the steps subsequent to fertilization raises the possibility that the m a t - idiomorph controls post-fertilization steps affecting both the m a t - and m a t + nuclei when they are together in the fertilized female organ. Since the S a l I - E e o R V fragment confers the Mat + phenotype, the 3' end of F P R 1 is dispensable for fertilization functions (Fig. 2C). Nevertheless, fertility is improved when the fragment extends beyond the SalI site. The additional sequence contains the carboxy-terminal part of the F P R 1 coding sequence. The poor fertility obtained with the S a l I - E c o R V fragment could be due to lower stability of the truncated F P R 1 m R N A or the encoded polypeptide relative to the normal m R N A or gene product. The reduced fertility may be due also to the disruption of the possible gene identified in the noncoding strand of F P R 1 . A sterile strain lacking the entire m a t + idiomorph is now available (S. Arnaise, personal communication). Only functions related to sexual reproduction are impaired in this strain, which displays no particular phenotype besides sterility. Use of this sterile strain as recipient in transformation assays will facilitate the molecular dissection of the m a t idiomorphs and will also allow the determination of the functions of m a t segments in the absence of interaction with a resident m a t idiomorph. Now it seems clear that m a t idiomorphs not only control the initiation of the sexual cycle but also fruiting body maturation. However cytological studies are necessary to identify those steps between fertilization and ascospore maturation that are submitted to m a t control. Acknowledgements. The two authors have contributed equally to the present work. We t h a n k Marguerite Picard and our colleagues for discussions and constant advice throughout this work. We are grateful to Louise Glass, Jeff Grotelueschen and Bob Metzenberg, Chuck Staben and Charles Yanofsky for communicating results prior to publication. We t h a n k Bob Metzenberg and Louise Glass for fruitful discussions during the sixteenth Fungal Genetic Conference at Asilomar (1991). We are very much indebted to Claude Gerbaud who set up and streamlined the computer software which were used for the protein comparisons. We are grateful to Joel B6gueret for providing us with the plasmid pM33-18M3. This work was supported by grants from the Institut National de la Sant6 et de la Recherche Mbdicale (grant 89009) and from the Fondation pour la Recherche M~dicale.

121 References

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Note added in proof. The nucleotide sequence of F M R 1 and FPR1 was deposited in the EMBL Data library, accession number X64194 and X64195 respectively.

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