commune contains a pheromone receptor gene and putative ... - NCBI

The EMBO Journal vol.14 no.2 1 pp.5271-5278, 1995

The mating-type locus Bal of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes J.Wendland, L.J.Vaillancourt", J.Hegner, K.B.Lengeler, K.J.Laddison1'2, C.A.Specht3, C.A.Raper' and E.Kothe4 Philipps-Universitat, Fachbereich Biologie-Molekulargenetik, 35032 Marburg, Germany, 'L.P.Markey Center for Molecular Genetics, Stafford Building, University of Vermont, Burlington, VT 05405 and 3Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307, USA 2Present address: Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA

4Corresponding author

Analysis of the multispecific Ba mating-type locus of Schizophyllum commune provided evidence that pheromones and pheromone receptors govern recognition of self versus non-self and sexual development in this homobasidiomycetous fungus. Four subclones of an 8.2 kb genomic fragment carrying Bacl specificity induced B-regulated sexual morphogenesis when introduced into a strain with one of the eight compatible Ba specificities that are known to exist in nature. One of these clones, which activated all other Ba specificities, contains a gene termed bar]. The predicted protein product of bar), as well as that of bar2, a homologous gene isolated from a Ba2 strain, has significant homology to known fungal pheromone receptor proteins in the rhodopsin-like superfamily of G protein-linked receptors. The other three active Bal clones were subcloned further to identify the minimal active element in each clone. Every active subclone contains a putative pheromone gene ending in a signal for possible isoprenylation. A message of .600 bp was observed for one of these genes, bapl(1). This paper presents the first evidence for a system of multiple pheromones and pheromone receptors as a basis for multispecific mating types in a fungus. Keywords: basidiomycete/development/G protein-coupled receptor/mating type/pheromones

Introduction Cell-to-cell communication is essential for development in all multicellular organisms and has long been a subject of particular interest in filamentous fungi with multiple mating types. The molecular mechanisms by which mating-type genes function to distinguish self from nonself and initiate sexual development are only partly understood. The genetically tractable homobasidiomycete Schizophyllum commune has served as a model in efforts to understand these mechanisms (for reviews see Raper, 1966; Raper, 1983, 1988; Novotny et al., 1991). The master regulators of self versus non-self recognition in S.commune are located in two unlinked genetic complexes © Oxford University Press

called A and B which govern separate and complementary pathways of sexual development culminating in fruit-body formation and meiosis. Each complex is composed of two linked loci: Aa and AP, with nine and 32 specificities, respectively, and Ba and BP, with nine specificities each (Raper et al., 1958a,b; Koltin et al., 1967; Stamberg and Koltin, 1973). The two A mating-type loci function independently but redundantly to regulate conjugate nuclear division and hook-cell formation. The two B loci independently but redundantly regulate nuclear migration and hook-cell fusion. Each haploid genome carries a specificity at each of these four loci which, in combination, define a particular mating type. The mating-type genes initiate intracellular events: cell fusions between hyphae occur regardless of mating type. The minimal requirement for full compatibility between haploid mates is a difference in specificity at either Aa or AP and a difference at either Ba or BP. A fully compatible mating results in the bilateral migration of nuclei from one mate into and throughout the other to form a morphologically distinct dikaryon (Figure 1). Each cell in the dikaryon contains two haploid nuclei, one from each mate, and cells are connected by a fused hook-cell at each septum. This is termed the A-on/B-on phenotype. Nuclear migration is established in semicompatible heterokaryons resulting from a mating in which there is a difference at one or both of the B loci but identity at the A mating-type loci. These A-off/B-on heterokaryons display a characteristic 'flat' morphology with irregularly branched, submerged hyphae, few aerial hyphae, and nuclei migrating continously from cell to cell accompanied by septal dissolution and reformation. In ascomycetous fungi, alternative versions of a single locus determine only two mating types. Many heterobasidiomycetes, such as Ustilago maydis, have two matingtype loci, one biallelic and one multiallelic. Outbreeding potential is therefore increased, but there are still

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Fig. 1. Regulation of sexual morphogenesis by the A and B matingtype loci of Scommune. Hyphal fusion occurs regardless of the mating types of paired strains; the recognition of self versus non-self occurs only after cell fusion. Development leading to fruiting and meiosis occurs only when two mates differ at both A and B mating-type loci (AxBxXAyBy). Fertilizing haploid nuclei migrate reciprocally from one mate into and throughout the other. In this illustration, the solid nucleus is resident and the hollow nucleus is migrating from a compatible mate through disrupted septa. A dikaryon with two nuclei of opposite types is established after the invading nucleus reaches an apical cell and pairs with a resident nucleus. This is followed by hookcell formation, conjugate division of the paired nuclei-one into the hook-cell, the other along the hyphal axis-hook-cell septation and hook-cell fusion. Repetition of this cycle maintains a 1:1 ratio of the two nuclear types in every cell at each cell division. The first and last events, nuclear migration and hook-cell fusion, are regulated by a difference in specificity of the B mating-type loci (AxBxXAxBy). The intervening events are regulated by a difference in specificity of the A mating-type loci (AxBxXAyBx). The former results in an A-off/B-on phenotype characterized by septal disruption, continuous nuclear migration, irregular branching and flat, submerged growth. The latter results in an A-on/B-off phenotype characterized by binucleate apical cells but uninucleate subterminal cells with uninucleate, unfused hookcells at each septum.

In the ascomycetous yeast Saccharomyces cerevisiae, recognition between the two mating types and subsequent sexual differentiation occurs via a pair of pheromones and pheromone receptors (for reviews see Kurjan, 1992; Whiteway and Errede, 1993). The pheromone from one mating type binds to the receptor of the other to initiate an intracellular signal transduction cascade beginning with the dissociation of a heterotrimeric G protein bound to the receptor. The two pheromone receptors, known as Ste2 and Ste3, are members of the rhodopsin-like superfamily of G protein-linked receptors, characterized by seven hydrophobic transmembrane domains. The pheromones, known as a-factor and a-factor, are processed to small peptides (12 and 13 amino acids in length, respectively) which are secreted from the cells. In U.maydis, each of the two specificities of the a mating-type locus consists of two genes, one coding for a pheromone and one for a pheromone receptor. Together these genes regulate cell-to-cell recognition, cell fusion and maintenance of filamentous growth (Banuett and Herskowitz, 1989; Froeliger and Leong, 1991; Bolker et al., 1992; Spelling et al., 1994). The two receptors, encoded by pral and pra2, are similar to the pheromone receptors of S.cerevisiae and also belong to the rhodopsinlike superfamily. Translation of the U.maydis mfal and mfa2 genes results in pheromone precursors of 40 and 38

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amino acids, respectively, each ending in a C-a-a-X signal motif for farnesylation, a motif which also occurs in the a-factor pheromone of S. cerevisiae. Transformation experiments indicate that the pheromone from one mating type of U.maydis activates the receptor of the opposite mating type, but is unable to activate the receptor of its own mating type (Bolker et al., 1992). This is analagous to the pheromone-receptor interaction in S.cerevisiae. Until now, detection of such mating-type systems has been confined to the ascomycetes and heterobasidiomycetes, in which only two different pheromone-receptor pairs function to distinguish two mating-type specificities and initiate cell fusion (Bolker and Kahmann, 1993). In this paper evidence is presented for a multiallelic system of pheromones and pheromone receptors governing recognition of self versus non-self to initiate a pathway of intracellular events in the homobasidiomycete S. commune.

Results B-regulated development is induced in cells transformed with the cloned Bal mating type locus The Bal mating-type specificity of S.commune was cloned on a 28 kb fragment of genomic DNA (Specht, 1995). This fragment activates B-regulated development (B-on) in strains representing all of the other eight Ba specificities when integrated via DNA-mediated transformation into their genomes. It did not activate Bal strains. Activity was signified by the transformation of regenerated protoplasts from fluffy mycelium with uninucleate cells, intact septa and many aerial hyphae, typical of an unmated homokaryon, to a 'flat' mycelium with stubby, irregular branches, broken septa, weakened cell walls and nuclei migrating continuously from cell to cell, typical of an Aoff/B-on heterokaryon (Figure 2). In test matings, flat (A-off/2B-on) transformants were capable of donating migrating nuclei to strains containing the same B specificity as the transformation recipient cells and a different A specificity. A fruiting dikaryon with fused hook-cells was subsequently established. Thus, both aspects of Bregulated development (i.e. nuclear migration and hookcell fusion) were activated by insertion of the transforming Ba] DNA into the genomes of strains with oher Ba specificities. The Bal locus consists of several active elements Subclones representing the entire Bal cosmid insert were tested for activity in transformation experiments. Four of the subclones, contained within a contiguous 8.2 kb fragment of DNA, activated differing spectra of haploid strains carrying the other eight Ba specificities (Figure 3). None of the subclones activated Ba] strains. Clone pBal11 transformed four of the other eight specificities; pBal12 activated only one other Ba specificity; pBal-13 was inactive; pBal-21 activated six of the eight specificities; and pBal-22 activated all of the other eight specificities. The subclones differed not only in their activation spectra, but also in the degree of response they elicited. Three of the four induced relatively weak flat reactions (+), while pBal-22, the clone capable of activating the full spectrum of different Ba specificities, produced very strong flat

Schizophyllum mating-type locus Bal

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Fig. 2. Comparative morphological phenotypes of transformants. Microscopic views at X300 magnification are shown at left; macroscopic views are shown at right. All cultures were inoculated simultaneously on CYM agar medium. The photographs at the top illustrate a strain of Ba2 mating type transfected with vector DNA only. The middle photographs illustrate the same strain transfected with DNA containing the bapl(J) pheromone gene which elicited a weak flat (+) B-on response. The photographs at bottom illustrate the same strain transfected with the bar] pheromone receptor gene which elicited a strong flat (+++ +) B-on response.

reactions (+++ ). Representative illustrations of these quantitative differences are shown in Figure 2. The Bal locus contains a pheromone receptor gene and putative pheromone genes The entire 8.2 kb active region of Bal was sequenced (EMBL accession number X77949 and GenBank accession number U32677). Sequence analysis revealed four open reading frames (ORFs) of particular interest. The largest protein deduced from the sequence is contained in the pBal-22 subclone, which elicits a strong (+ + +) flat reaction in transformants (see Figure 3). Sequence analysis of the cDNA corresponding to this ORF confirmed the existence of five small introns interrupting the ORF. The predicted protein product is significantly homologous to several known fungal pheromone receptors (Figure 4). This gene was named bar], for B alpha receptor 1. A hydrophobicity plot of the predicted Barl protein reveals seven hydrophobic, possible membrane-spanning domains which are characteristic of the rhodopsin-like superfamily of G protein-linked receptors.

The predicted amino acid sequence of Barl shares another feature with most members of this family: two cysteine residues are conserved at the predicted extracellular face of the third transmembrane domain (111:0) and in the second extracellular loop (IV-V) (Figure 4). These cysteines are thought to form a disulfide bridge stabilizing the secondary structure of the molecule (Baldwin, 1994). No other ORFs having significant similarity to receptor genes, or to any other sequences in the databases, could be identified within the active 8.2 kb Bal fragment. The presence of a pheromone receptor gene prompted a search for potential pheromone genes within the Bal locus. Known fungal pheromone genes are small and they often encode a C-a-a-X signal for farnesylation at the Cterminus of the protein (Bolker and Kahmann, 1993; Duntze et al., 1993). Three ORFs encoding predicted proteins of 50 amino acids or less and ending in a possible isoprenylation signal motif were identified as putative pheromone genes within the 8.2 kb active region (Figures 3 and 5). One of the putative pheromone genes, bapl(l) (for B alpha 1 pheromone), is preceded by seven direct

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Barl Bar2 pral pra2 Ste3

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Barl Bar2 pral pra2 Ste3

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S-----P--L-I ---VA---M-V--II-KBar2 ----T--A--------I-CCLV pral M-DHIT-F--LVA-F-VLM-FA--IKSK-V-LIMLSI-LM--N-DN-V--MV-WK pra2 MFSGKENVS-GVLCL-AGCISTSSCLI----K-I-VLLM-F-CFT-LV-KG--AL-FNN

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V-A-LV-RW-NLR-R--TAA-SAQ--G--QKK-F-IF---IC-RVLVSAGQFY--IQSL V-ATLV-FV-YXK-KDVRDI--CTN-G-NLT-FA--LIFCFIIILVMF-FSVYTF-QDL TM 6

Barl KTENIQPWVSLAVTHYGFGRIDQVPAIVWLSQHLIVVCNELTRWCAPVSAFIFFFYFGF Bar2 -AKKVS-Y--W ------ Y-I--------I-R-NR-L-ASY-----SS-AI-L--------

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Fig. 3. Activity spectra of subclones of the 8.2 kb Bal mating-type locus. Restriction enzyme sites are as follows: E = EcoRI, P = PstI, S = SacI, H = HindIII, K = KpnI. Location and direction of transcription of possible pheromone genes bapl(1), bapl(2) and bapi(3) and the pheromone receptor gene, bar], are shown above the clones. Activity of each clone was scored either as a weak (+) or strong (+++ +) B-on phenotype (see Figure 2) when used as the transfecting DNA in strains of all nine Ba specificities listed at left. (-) Indicates no activity detected.

21 bp repeats which have homology to similar repeats found upstream of the U.maydis pheromones mfal and mfa2. Comparable repeated sequences were not found near any of the other putative pheromone genes. A 452 bp restriction fragment containing only the direct repeats and the adjacent bap(l) ORF was tested for activity in transformation. This small fragment was sufficient to induce B-regulated development when integrated into BaX2 and Boc3 strains (Figure 6). Expression of bapl(J) was shown by Northern blot analyses (Figure 7). A second putative pheromone gene, bapl(2), was active in transformation when subcloned to a small restriction fragment of 466 bp. The active subclone pBaxl-1l contains a third putative pheromone gene, bapi(3) located 40 bp downstream of an EcoRI restriction site. Subclones resulting from digestion with EcoRI did not activate Bregulated development, whereas a PstI subclone of 932 bp containing bapi(3) and -200 bp of possible promoter sequences was active in transformation (Figure 6).

Transformants containing different Bal subclones behave differently in mating tests In transformation experiments, the four active Bcl subclones induced two types of B-regulated development: a very strong reaction type was induced by the subclone containing bar], while the other three active subclones consistently induced a weaker flat reaction (+ + + versus +, respectively, Figures 2 and 3). Strains transformed with subclones were tested in matings. Strains of either Bcx2 or BocS specificities were transformed with bapl(J) (+ reaction type), bar] (+ + + reaction type) or both on a larger subclone (+ + + reaction type, indistinguishable from that induced by bar] alone; Figure 3). Significant differences were seen when the various Bon transformants were mated with testers of the same B specificity as the transformation recipient and a different A specificity. All transformants containing bapl(J), 5274

pra2 QIGGLL-YT-W-EV-TN-N--LF--VDTIA Ste3 QQVEGHYTFKNTHSSTIWNT-IKFDPGRPI Barl Bar2 pral pra2 Ste3

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Fig. 4. Alignment of Barl and Bar2 with pheromone receptors pral and pra2 of Umaydis and Ste3 of S.cerevisiae. The C-termini of Barl, Bar2 and Ste3 are truncated to the length of the pral and pra2 receptors. No homology is seen between the deleted C-terminal parts of Ste3 and the Scommune receptors. Potential transmembrane domains are indicated (TM1 through TM7).

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bapl(2) or bapl(3) were capable of donating migrating nuclei to the tester. The process of nuclear migration was relatively slow, however, requiring a period of 7 days at 30°C after mating. Patchy dikaryons with some unfused hook-cells were established at the end of this time. The dikaryons fruited and sporulated normally. Transformants containing both bar] and bapl(J) donated nuclei to the testers and quickly established vigorous dikaryons with uniformly fused hook-cells throughout the tester mycelium. Formation of the dikaryon, which went on to fruit and sporulate normally, was completed in 3 days, significantly faster than the 7 days required for transformants containing only bapl(J). In contrast, strains transformed only with bar] were completely incapable of donating migrating nuclei to the testers. Heterokaryons did eventually form where fusion occurred along the junction of the two mates. When these heterokaryons were subcultured under selective conditions, they did eventually develop (after a period of several

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weeks) into dikaryons with fused hook-cells which fruited and sporulated. All transformants had the flat, A-off/B-on morphology. Since the nuclear migration pathway is already activated via transformation, invading nuclei cannot superimpose an organized process of polarized migration within the transformant in backcrosses to a tester strain. Thus, none of the transformed strains accepted migrating nuclei in test matings.

Comparison of barl with bar2, a Ba2 pheromone receptor gene Southern hybridization experiments were performed using the bar] cDNA to probe genomic DNA from various S.commune strains representing other Ba specificities. Sequences homologous to bar] were detected in all of these strains (data not shown). A hybridizing fragment isolated from a partial library of a Ba2 strain was active in transformation (+ + +) in a Ba3 strain, but inactive in Ba2, as would be expected for a Ba2 pheromone receptor gene bar2. Sequence analysis confirmed strong homology of bar2 to bar]. Divergency was evident in extracellular loops of the serpentine receptors which are thought to be involved in pheromone binding (Figure 4) (Sen and Marsh, 1994). The C-termini presumably coupling the receptor to the signal transduction pathway show strongest similarity, which would be expected for intracellular factor binding sites (Marsh et al., 1991).

Discussion Analysis of the Ba] mating-type locus of S.commune revealed four regions within an 8.2 kb DNA fragment, each of which was capable of eliciting a Bal specific

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B

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2200 bp

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mating-type response when integrated into the genomes of other Ba specificities. One of these active regions contains a gene, barl, which encodes a protein with strong similarities to pheromone receptor genes in U.maydis and S.cerevisiae. The Ba2 mating type contains a homologous but distinct pheromone receptor gene, bar2, implying that each Ba specificity contains at least one different matingtype-specific bar gene. In addition to bar], three other elements within the Bal locus were capable of activating B-regulated development. Each of the active regions contains an ORF predicted to encode a small peptide with a putative C-terminal C-XX-X isoprenylation signal. These ORFs were designated

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as putative pheromone genes by analogy to similar genes encoding small farnesylated peptide pheromones in several other fungi (Clarke, 1992; Duntze et al., 1993). The existence of other pheromone genes that do not meet these criteria has not been ruled out. Evidence that these genes may encode functional pheromones is provided by Northern blot analysis showing that bapl(J) is transcribed, and by transformation experiments demonstrating that minimal restriction fragments containing only the bap](]), bap](2) or bap](3) ORFs and some upstream sequences activate B-regulated development. The putative pheromones activate only a partial spectrum of the other Ba specificities. In contrast, the pheromone receptor Barl elicits a very strong B-on phenotype (± ++) in all eight other Ba specificities. The single inactive subclone within the 8.2 kb active region of Bal (pBa-13, Figure 3) contains no potential pheromone gene. The different degrees of activation of the B-on phenotype by the putative pheromones versus the Barl receptor may be due to an autoregulatory effect. Interactions between receptors and pheromones in some other fungi result in up-regulation of both types of molecules (Marsh et al., 1991; Spelling et al., 1994; Whiteway and Errede, 1993). Sequences required for similar up-regulation in S.commune may not be included on the three active Bal subclones containing only pheromone genes. Considerable redundancy was evident in the interactions between Bal and the various other Ba specificities. For example, all four active Bal subclones induced Bregulated development in Ba2 strains. Only a single pheromone receptor gene, bar2, was isolated from the Ba2 mating type. These data imply that different Bal pheromones can activate a single Ba2 receptor. This is in contrast to all other known fungal pheromone-receptor systems in which each receptor can be activated by only one pheromone. In S.commune, the evidence also suggests that individual pheromones are able to activate more than one receptor. This was demonstrated by transformation of small restriction fragments containing either bapl(l) or bapl(3) into Ba2 or Ba3 strains, both of which were activated as a result. This also contrasts with known fungal pheromone-receptor systems in which each pheromone can activate only a single receptor. Qualitative differences in the developmental effects of the subclones containing pheromone genes compared with the subclone containing bar] provide a clue about the nature and function of the molecules encoded by these genes. Strains transformed with bar] strongly express the B-on phenotype in which nuclei migrate continuously from cell to cell (+++ + type, Figures 2 and 3), yet are completely incapable of donating migrating nuclei to a partner in test matings. In contrast, the same strains transformed with pheromone genes express a relatively weak B-on phenotype (+ type, Figures 2 and 3), yet are capable of donating migrating nuclei in comparable test matings. This suggests that Barl and the pheromones play different roles in the initiation of B-regulated nuclear migration. The difference is consistent with the concept that bar] encodes a non-diffusible, membrane-bound receptor molecule, while the pheromone genes encode small peptides that can diffuse to adjacent cells. The data presented here suggest the following model for the action of the Ba genes. During mating, pheromone(s)

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encoded by the Ba genes of one specificity in the fertilizing nucleus (endogenous or genes introduced by transformation) are secreted and act as advance signals that activate membrane-bound receptor molecules encoded by the resident nucleus of a different specificity in nearby cells. These cell(s) are thus prepared for invasion by undergoing septal breakdown to permit passage of the fertilizing nucleus; in the absence of a signal, the septa remain intact and continue to act as barriers to the passage of nuclei from one cell to the next. The process of nuclear migration between two haploid homokaryons with compatible pheromones and pheromone receptors is normally bidirectional, resulting in the rapid conversion of each mate to a heterokaryon with two types of nuclei. We propose that reciprocal nuclear migration in S.commune is controlled via pheromones and pheromone receptors encoded within the Ba locus. In previously described fungal pheromone-receptor systems, alternative pairs of pheromones and receptors distinguish only two different mating specificities. There are nine Ba specificities in Schizophyllum, requiring each receptor to distinguish at least eight different non-self pheromones from those encoded by the self specificity. The existence of three different active pheromone genes within Bal means that the number of pheromones that must be recognized may be even greater. In addition the BPl locus, which works independently but redundantly to activate B-regulated development and is genetically distinct from Ba, also contains putative pheromones and pheromone receptors (unpublished results). This suggests that Ba receptors not only distinguish among the Ba pheromones, but also do not respond to pheromones encoded by the BP loci. According to precedent, compatible pheromonereceptor interactions result in the intiation of a G proteinmediated signal transduction cascade, which in turn activates many different genes involved in the B-regulated development. Evidence for numerous genes involved in this pathway regulating B-dependent development has been obtained by previous mutational analyses (Raper and Raper, 1964, 1966, 1973). Some of these mutants may be useful for the molecular characterization of the cascade of events initiated by the B mating-type genes. The study reported here provides the beginning of an understanding of how the B mating-type genes function to initiate these events.

Materials and methods Fungal and bacterial strains, culturing conditions and transformation of Scommune The Scommune strains were all derived from those originally collected and identified by J.R.Raper and associates at Harvard University (Raper et al., 1958b; Koltin et al., 1967). Those used in transformation studies included all of the Bax specificities known to exist in nature. Transformation (Mufioz-Rivas et al., 1986a; Specht et al., 1991) was performed using the trpl (Mufioz-Rivas et al., 1986b) and ural (Froeliger et al., 1987) genes as selectable markers. The selective marker trpl was incorporated into each strain by outcrossing to a trpl strain. Ba specificities of the auxotrophic strains used in the transformation studies and their respective strain designations are as follows. Bal: H4-40, Tl 1, V115-3 and V121-10. Ba2: TI and T26. Bo3: T2, V113-17 and V12420. Ba4: V131-5 and V131-12. Ba5: V112-3. Ba6: V123-5. Ba7: V12520 and V119-32. Bac8 (formerly Bal'): V117-13 and V117-24. BCa9 (formerly Bac2'): V118-4 and V118-8. Culturing techniques and media

Schizophyllum mating-type locus Bac1 used were those described by Raper and Hoffman (1974). Scommune DNA was extracted as described (Lengeler and Kothe, 1994). Escherichia coli K12 DHSa and plasmids pT7T3al8 and pUC18 (Life Technologies, Gaithersburg, MD, USA) were used for cloning.

Expression and sequence analyses A bar] cDNA was amplified by using the PCR and primers deduced from the translational start site (ATGCTGGACCCGCTCTACCCG) and complementary to a sequence from the intracellular, C-terminal part of the receptor gene (AGGTGAGTTCGGAGGTGAAGT). AMV reverse transcriptase (Boehringer, Mannheim) was used to transcribe 10 ig of total RNA from which the cDNA was amplified with both primers. The product was shorter than the respective fragment from the subcloned DNA by -250 bp. The PCR product was cloned and sequenced to locate five small introns disrupting the ORF. Isolation of a 5 kb PstI restriction fragment carrying bar2 from a partial library was achieved by colony hybridization. Sequencing was performed using automated cycle sequencing (Applied Biosystems, Weiterstadt and dsDNA Cycle Sequencing System kit, Life Technologies, Gaithersburg, MD, USA) or 35S sequencing (T7 polymerase sequencing kit, USB Bad Homburg, Germany). Data were analyzed with BLAST and MacVector software packages and have been deposited at the EMBL data bank (accession numbers X77949 and X91168) and GenBank (accession number U32677). Northern and Southern hybridization experiments were performed according to protocols given by Lucas et al. (1977) and Sambrook et al. (1989). Exposure times varied between 4 and 10 days.

Acknowledgements The authors thank Albrecht Klein for fruitful discussion; Luc Giasson and Robert Ullrich for their support and for use of the genomic library which was used to isolate Bal; Brian Foley, Tom Fowler, Zafer Hatahet and Benjamin Weinstock for technical advice and assistance. E.K., J.W., J.H. and K.B.L. were supported by the Deutsche Forschungsgemeinschaft (grants KolO89/2-1 and KolO89/2-2 to E.K. and grants of the Graduiertenkolleg Enzymchemie to J.W. and J.H.). K.J.L., L.J.V. and C.A.R. were supported by the National Institutes of Health (GM15292 to L.J.V. and C.A.R.) and by the National Science Foundation (MCB9205633 to C.A.R.). C.A.S. was supported by the National Institutes of Health (GM31318 to P.W.Robbins and CA14051 to R.O.Hynes).

References Baldwin,J.M. (1994) Structure and function of receptors coupled to G proteins. Curr Opin. Cell Biol., 6, 180-190. Banuett,F. and Herskowitz,I. (1989) Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis. Proc. Natl Acad. Sci. USA, 86, 5878-5882. Bolker,M. and Kahmann,R. (1993) Sexual pheromones and mating responses in fungi. Plant Cell, 5, 1461-1469. Bolker,M., Urban,M. and Kahmann,R. (1992) The a mating-type locus of U. maydis specifies cell signaling components. Cell, 68, 441-450. Clarke,S. (1992) Protein isoprenylation and methylation at carboxylterminal cysteine residues. Annu. Rev. Biochem., 61, 355-386. Duntze,W., Betz,R. and Popping,B. (1993) Lipopeptide pheromones of yeast and basidiomycetes involved in mating. In Kurjan,J. and Taylor,B.L. (eds), Signal Transduction: Prokaryotic and Simple Eukaryotic Systems. Academic Press, San Diego, CA, pp. 239-254. Froeliger,E.H. and Leong,S.A. (1991) The a mating-type alleles of Ustilago maydis are ideomorphs. Gene, 100, 113-122. Froeliger,E.H., Mufioz-Rivas,A.M., Specht,C.A., Ullrich,R.C. and Novotny,C.P. (1987) The isolation of specific genes from the basidiomycete Schizophyllum commune. Curr Genet., 12, 547-554. Giasson,L., Specht,C.A., Milgrim,C., Novotny,C.P. and Ullrich,R.C. (1989) Cloning and comparison of Aa mating-type alleles of the basidiomycete Schizophyllum commune. Mol. Gen. Genet., 218, 72-77. Gillissen,B., Bergemann,J., Sandmann,C., Schoeer,B., Bolker,M. and Kahmann,R. (1992) A two-component regulatory system for self/nonself recognition in Ustilago maydis. Cell, 68, 647-657. Koltin,Y., Raper,J.R. and Simchen,G. (1967) The genetic structure of the incompatibility factors of Schizophyllum commune: the B factor. Proc. NatI Acad. Sci. USA, 57, 55-62. Kues,U. and Casselton,L.A. (1993) The origin of multiple mating-types in mushrooms. J. Cell Sci., 104, 227-230.

Kuies,U., Richardson,W.V.J., Tymon,A.M., Mutasa,E.S., Gottgens,B., Gaubatz,S., Gregoriades,A. and Casselton,L.A. (1992) The combination of dissimilar alleles of the Aa and AO gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus. Genes Dev., 6, 568-577. Kurjan,J. (1992) Pheromone response in yeast. Annu. Rev. Biochem., 61, 1097-1129. Lengeler,K. and Kothe,E. (1994) Molecular characterization of ural, a mutant allele for orotidine-5 '-monophosphate decarboxylase in Schizophyllum commune. FEMS Microbiol. Lett., 119, 243-248. Lucas,M.C., Jacobsen,J.M. and Giles,N.H. (1977) Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa. J. Bacteriol., 130, 1192-1198. Marsh,L., Neiman,A.M. and Herskowitz,I. (1991) Signal transduction during pheromone response in yeast. Annu. Rev. Cell Biol., 7, 699-728. Mufnoz-Rivas,A., Specht,C.A., Drummond,B.J., Froeliger,E., Novotny, C.P. and Ullrich,R.C. (1986a) Transformation of the basidiomycete Schizophyllum commune. Mol. Gen. Genet., 205, 103-106. Mufnoz-Rivas,A.M., Specht,C.A., Ullrich,R.C. and Novotny,C.P. (1986b) Isolation of the DNA sequence coding indole-3-glycerol phosphate synthetase and phosphoribosylanthranilate isomerase of Schizophyllum commune. Curr Genet., 10, 909-913. Novotny,C.P., Stankis,M.M., Specht,C.A., Yang,H., Giasson,L. and Ullrich,R.C. (1991) The Aa mating-type locus of Schizophyllum commune. In Bennett,J.W. and Lasure,L.L. (eds), More Gene Manipulations in Fungi. Academic Press Inc., San Diego, CA, pp. 235-257. Raper,C.A. (1983) Controls for development and differentiation in basidiomycetes. In Bennett,J.W. and Ciegler,A. (eds), Secondary Metabolism and Differentiation in Fungi. Marcel Dekker, New York, pp. 195-238. Raper,C.A. (1988) Schizophyllum commune: a model for genetic studies of the basidiomycetes. In Ingram,D.S. and Williams,P.H. (eds), Advances in Plant Pathology: Genetics of Plant Pathogenic Fungi. Academic Press, London, Vol. 6, pp. 511-522. Raper,C.A. and Raper,J.R. (1964) Mutations affecting heterokaryosis in Schizophyllum commune. Am. J. Bot., 51, 503-513. Raper,C.A. and Raper,J.R. (1966) Mutations modifying sexual morphogenesis in Schizophyllum. Genetics, 54, 1151-1168. Raper,C.A. and Raper,J.R. (1973) Mutational analysis of a regulatory gene for morphogenesis in Schizophyllum. Proc. Natl Acad. Sci. USA, 70, 1427-1431. Raper,J.R. (1966) Genetics of Sexuality in Higher Fungi. Ronald Press, New York. Raper,J.R. and Hoffman,R.M. (1974) Schizophyllum commune. In King,R.C. (ed.), Handbook of Genetics. Plenum Press, New York, pp. 597-626. Raper,J.R., Baxter,M.G. and Middleton,R.B. (1958a) The genetic structure of the incompatibility factors in Schizophyllum commune. Proc. Natl Acad. Sci. USA, 44, 889-900. Raper,J.R., Krongelb,G.S. and Baxter,M.G. (1958b) The number and distribution of incompatibility factors in Schizophyllum commune. Am. Nat., 92, 221-232. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schulz,B., Banuett,F., Dahl,M., Schlesinger,R., Schafer,W., Martin,T., Herskowitz,I. and Kahmann,R. (1990) The b alleles of U. maydis, whose combinations program pathogenic development, code for polypeptides containing a homeodomain-related motif. Cell, 60, 295-306. Sen,M. and Marsh,L. (1994) Noncontiguous domains of the a-factor receptor of yeasts confer ligand specificity. J. Biol. Chem., 269, 968-973. Specht,C.A. (1995) Isolation of the Ba and BP mating-type loci of Schizophyllum commune. Curr Genet., in press. Specht,C.A., Mufioz-Rivas,A., Novotny,C.P. and Ullrich,R.C. (1991) Transformation of Schizophyllum commune: an analysis of specific properties. Exp. Mycol., 15, 326-335.

Specht,C.A., Stankis,M.M., Giasson,L., Novotny,C.P. and Ullrich,R.C. (1992) Functional analysis of the homeodomain-related proteins of the Aa locus of Schizophyllum commune. Proc. Natl Acad. Sci. USA, 89, 7174-7178. Spelling,T., Bolker,M., Lottspeich,F., Frank,R.W. and Kahmann,R. (1994) Pheromones

trigger filamentous growth

J., 13, 1620-1627.

in

Ustilago maydis.

EMBO

5277

J.Wendland et a!. Stamberg,J., and Koltin,Y. (1973) The organisation of the incompatibility factors in higher fungi: the effect of structure and symmetry on breeding. Heredity, 30, 15-26. Stankis,M.M., Specht,C.A., Yang,H., Giasson,L., Ullrich,R.C. and Novotny,C.P. (1992) The Aa mating-type locus of Schizophyllum commune encodes two dissimilar multiallelic homeodomain proteins. Proc. Natl Acad. Sci. USA, 89, 7169-7173. Whiteway,M. and Errede,B. (1993) Signal transduction pathway for pheromone response in Saccharomyces cerevisiae. In Kurjan,J. and Taylor,B.L. (eds), Signal Transduction: Prokaryotic and Simple Eukaryotic Systems. Academic Press Inc., San Diego, CA, pp. 189-237. Received on February 22, 1995; revised on August 17, 1995

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