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Molecular Characterization of tol, a Mediator of Mating-Type-Associated Vegetative Incompatibility in Neurospora crassa


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Copyright1999 by the Genetics Society of America

Molecular Characterization of


, a Mediator of Mating-Type-Associated

Vegetative Incompatibility in

Neurospora crassa

Patrick Ka Tai Shiu and N. Louise Glass

The Biotechnology Laboratory and The Botany Department, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Manuscript received July 20, 1998 Accepted for publication October 12, 1998


The mating-type locus in the haploid filamentous fungus, Neurospora crassa, controls mating and sexual development. The fusion of reproductive structures of opposite mating type, A and a, is required to initiate sexual reproduction. However, the fusion of hyphae of opposite mating type during vegetative growth results in growth inhibition and cell death, a process that is mediated by the tol locus. Mutations in tol are recessive and suppress mating-type-associated heterokaryon incompatibility. In this study, we describe the cloning and characterization of tol. The tol gene encodes a putative 1011-amino-acid polypeptide with a coiled-coil domain and a leucine-rich repeat. Both regions are required for tol activity. Repeat-induced point mutations in tol result in mutants that are wild type during vegetative growth and sexual reproduction, but that allow opposite mating-type individuals to form a vigorous heterokaryon. Transcript analyses show that tol mRNA is present during vegetative growth but absent during a cross. These data suggest that tol transcription is repressed to allow the coexistence of opposite mating-type nuclei during the sexual reproductive phase. tol is expressed in a mat A, mat a, A/a partial diploid and in a mating-type deletion strain, indicating that MAT A-1 and MAT a-1 are not absolutely required for transcription or repression of tol. These data suggest that TOL may rather interact with MAT A-1 and/or MAT a-1 (or downstream products) to form a death-triggering complex.


NTRY into the sexual cycle in the filamentous fun- the sexual cycle, fusion of opposite mating-type hyphae during vegetative growth results in growth inhibition gus, Neurospora crassa, requires the fusion of

repro-ductive structures of opposite mating types (A and a). and cell death (Beadle and Coonradt 1944; Garn-jobst1953). At least 10 additional genetic determinants Cell fusion results in the presence of opposite

mating-type nuclei within a common cytoplasm. Proliferation called heterokaryon incompatibility (het) loci also re-strict formation of vegetative heterokaryons; genetic dif-of A and a nuclei occurs in the ascogenous hyphae

before formation of the crozier, where opposite mating- ferences at these loci are not required for fertilization nor do they interfere with sexual reproduction (for re-type nuclei are partitioned (Raju1980). Karyogamy

be-tween A and a nuclei occurs in the crozier and is immedi- view seeGlassandKuldau1992;Leslie 1993). The mat A and mat a sequences have been character-ately followed by meiosis and a single mitotic division

to form eight linearly arranged ascospores. Over 200 ized and are composed of dissimilar sequences (Glass et al. 1990; Staben and Yanofsky 1990). The 5301-progeny are typically formed from a single fertilization

event. Mating-type function is required for both cell bp A idiomorph contains three open reading frames (ORFs), mat A-1, mat A-2, and mat A-3. The mat A-1 gene fusion and for postfertilization events, presumably

dur-ing the proliferation and partitiondur-ing of opposite mat- determines mating identity and also confers mating-type-associated heterokaryon incompatibility (Glasset ing-type nuclei in the crozier (GriffithsandDelange

1978;Griffiths1982;GlassandLee 1992;Ferreira al. 1990). Strains that contain null mutations in mat A-1 are both sterile and heterokaryon compatible with a et al. 1998).

In addition to its role during the sexual cycle, the strains (Griffiths 1982; Saupe et al. 1996). MAT A-1 mating-type locus in N. crassa has a function during displays a region of similarity to the mating-type tran-vegetative growth. Although fusion of opposite mating- scriptional regulator, MATa1, of Saccharomyces cerevisiae type reproductive structures is required for entry into (Glass et al. 1990). The mat A-2 and mat A-3 genes are not required for mating identity or heterokaryon incompatibility, but are required for postfertilization This article is dedicated to Dr. Dorothy Newmeyer, who discovered functions (GlassandLee1992;Ferreira et al. 1996). tol.

The a idiomorph contains only one ORF, mat a-1.

Corresponding author: N. Louise Glass, The Biotechnology

Labora-A functional mat a-1 is required for mating identity, tory, Rm. 237 Wesbrook Bldg., The University of British Columbia,

Vancouver, BC, V6T 1Z3 Canada. E-mail: glass@unixg.ubc.ca heterokaryon incompatibility, and postfertilization


andBarry1977; RLM 04-08, Table 1), RLM 04-08 was crossed functions (Staben and Yanofsky 1990). As with mat

with FGSC (Fungal Genetics Stock Center, Kansas City, KS) A-1, mutations in mat a-1 result in strains that are sterile

strain 2226 (Mauriceville wild-type a). Numerous restriction and that are able to form vigorous heterokaryons with fragment length polymorphisms (RFLPs) have been observed either A or a strains (Griffiths and Delange 1978). between Mauriceville strains (FGSC 2225 and 2226; Table 1) and the Oak Ridge background of standard laboratory strains. An exceptional mat a-1 mutant, am33, is fertile but

hetero-Genomic DNA from ser-3 A progeny were screened for Mau-karyon compatible with A strains (Griffiths and

De-riceville LG IV by hybridization to cosmid G4:A9 (which

con-lange1978). The am33mutant contains a missense

muta-tains trp-4, a LG IV locus closely linked to tol ). A progeny, R5-tion in the carboxyl-terminal region of mat a-1 (Staben 28 (Table 1) had RFLP patterns identical to the FGSC 2226 andYanofsky1990). MAT a-1 has features in common parent when probed with G4:A9.

The strain NE-1 (his-5 tol trp-4 A) was constructed by crossing with transcriptional regulators, specifically an HMG

his-5 A (FGSC 456) with tol trp-4 a (FGSC 2337); a His2and box. The HMG box of MAT a-1 has been shown to bind

Trp2progeny was selected. Crossing of NE-1 to R1-09 (un-3 DNA and to be required for conferring mating identity,

a) gave R5-27 (un-3; his-5 tol trp-4 a). The presence of the tol2 but not for heterokaryon incompatibility (Philleyand allele (N83) in R5-27 was confirmed by crossing with RLM 04-Staben1994). 08 (Table 1); approximately half of the A/a partial diploid progeny displayed normal growth rates (see Figure 2). The A recessive mutation unlinked to the mating-type

lo-strain R5-27 was subsequently crossed with a pan-2-containing cus, tolerant (tol), suppresses mating-type-associated

het-strain (12-21-388) to give het-strains R4-71 and R4-72 (Table 1), erokaryon incompatibility such that tol A and tol a strains which were used for transformation assays.

form a vigorous heterokaryon (Newmeyer1970). Mat- Chromosome walk and physical mapping:A trp-4-containing ing activity of these strains is unaffected and tol strains cosmid was identified from the Orbach/Sachs pMOcosX ge-nomic library (Orbach1994; available from the FGSC) using show normal fertility in crosses with an opposite

mating-a trp-4 clone (Vollmer and Yanofsky1986) as a probe. A type strain. Attempts to identify additional suppressors

chromosome walk was performed in both directions using of mating-type-associated incompatibility resulted only end fragments as probes for each step. Selected cosmids were in the isolation of additional tol alleles (Vellani et al. physically mapped by RFLP analysis (Metzenberget al. 1985).

Transformation assay and subcloning of tol: Spheroplast 1994). The tol mutation apparently does not suppress

preparation and transformation were performed as previously incompatibility due to differences at other het loci (

New-described (Schweizer et al. 1981;Vollmer and Yanofsky meyer1970;LeslieandYamashiro1997).

1986) with the exception of selecting for resistance to hygro-This article reports the isolation and molecular char- mycin B (250 units/ml; Calbiochem). Cosmids in the walk acterization of the tol gene of N. crassa. The tol gene were assayed for Tol1activity by cotransforming the cosmids (conferring hygromycin resistance) with a mat A-1-containing encodes a putative 1011-amino-acid (aa) polypeptide

pOKE103 construct (pOKE103 has a pan-2 selectable marker; with a coiled-coil domain and a leucine-rich repeat.

J. GrotelueschenandR. L. Metzenberg,unpublished re-Mutants of tol obtained by repeat-induced point

muta-sults) into strain 71 (his-5 tol trp-4; pan-2 a) and strain R4-tion (RIP;Selker1990) lose their mating-type hetero- 72 (ade-3B; trp-4 cot-1; pan-2 A) spheroplasts. The tol gene karyon incompatibility function but are otherwise phe- was further subcloned into the hygromycin-resistant vector (pCB1004;Carrollet al. 1994). Homokaryotic transformants notypically normal during vegetative growth and sexual

were isolated according toEbboleandSachs(1990). reproduction. Expression studies indicate that tol is not

Nucleic acid isolation, DNA hybridization, and reverse

tran-transcriptionally repressed or activated by MAT A-1 scriptase PCR analysis: Standard molecular biology proce-and/or MAT a-1 during vegetative growth but is appar- dures were used throughout (Sambrooket al. 1989). Genomic ently repressed during sexual development. DNA isolation from Neurospora was adapted fromOakleyet al. (1987). Total RNA was extracted according toLogemann et al. (1987) and enriched for poly(A)1using the OligoTex mRNA kit (QIAGEN, Chatsworth, CA). Gel electrophoresis MATERIALS AND METHODS

and nucleic acids transfer to Nylon filters (Schleicher and

Strains, media, and culturing methods:The N. crassa strains Schuell, Keene, NH) were performed according to manufac-used in the study are listed in Table 1. Culturing and crossing, turer’s specifications. [a-32P]dCTP-labeled probes

(Amer-using Vogel’s (Vogel 1964) and Westergaard’s (Wester- sham, Oakville, ON) were generated from digested DNA using

gaard and Mitchell 1947) media, respectively, were per- the T7 QuickPrime Kit (Pharmacia, Baie d’Urfe, Quebec). formed as previously described, with modification (Davisand For reverse transcriptase (RT)-PCR, cDNAs were synthesized

Deserres1970;Perkins1986). For heterokaryon tests, 2-ml co- by the Not I-d(T)6primer using a procedure from the First

nidial suspensions (107/ml) of two strains containing different

Strand cDNA Synthesis Kit (Pharmacia). PCRs of DNA and auxotrophic markers were coinoculated onto vegetative cDNA were performed using primers tol 13 (59GGGCGGAG growth media (Vogel 1964). A compatible heterokaryon GATAGGAGG 39; bases 641 to 657) and tol 11 (59CCAGCAG forms a vigorously conidiating culture after 3 days of incuba- TGGCTCAGC 39; bases 1144 to 1129). The tol2mutant allele tion with a mean growth rate of 7 cm/day. Mating-type in- (N83) was amplified from strain R5-27 DNA using primers tol compatible heterokaryons are usually aconidial and have a ATG1 (59 CCTGGGCTCACCTATGC 39; base 250 to234) growth rate ofz0.7 cm/day (Vellaniet al. 1994). For perithe- and tol 39-end (59CGGCGGGATCTCTTTCTG 39; base 2894 cial RNA extraction, crosses were performed in petri dishes to 2877). The tol cDNA and tol2 (N83) PCR products were containing Westergaard medium layered with Miracloth mem- cloned into the PCRII vector using a TA cloning kit (In-brane (Calbiochem, La Jolla, CA). Perithecia were scraped vitrogen, San Diego) and subjected to DNA sequencing. The from the Miracloth and RNA was extracted as described below. entire DNA sequence of two different tol2(N83) clones was

Strain construction:To introgress part of Mauriceville link- determined and the mutation point was confirmed from three additional subclones.



Neurospora crassastrains

Strain Genotype Origin

his-5 A FGSC 456

R1-09 un-3 a R. L. Metzenberg

tol trp-4 a FGSC 2337

tol a FGSC 4142

NE-1 his-5 tol trp-4 A This study

MV 1c-a Mauriceville WT a FGSC 2226

MV 1 c-A Mauriceville WT A FGSC 2225

RLM 04-08 T(ILIIR) 39311 ser-3 A R. L. Metzenberg

R5-28 T(ILIIR) 39311 ser-3 A This study

(Mauriceville background on LG IV)

R5-27 un-3; his-5 tol trp-4 a This study

12-21-388 ad-3B al-2; cot-1; pan-2 A A. J. F. Griffiths

R4-71 his-5 tol trp-4; pan-2 a This study

R4-72 ad-3B; trp-4 cot-1; pan-2 A This study

74-OR23-1VA Oak Ridge WT A FGSC 2489

A2 ad-3A nic-2 cyh-1 a A. J. F. Griffiths

I-20-41 ad-3B arg-1; tol A A. J. F. Griffiths

I-20-26 ad-3B arg-1 A A. J. F. Griffiths

I-10-1 ad-3A nic-2; tol a A. J. F. Griffiths

I-1-51 ad-3A nic-2 a A. J. F. Griffiths

OR8-1a Oak Ridge WT a FGSC 988

RLM 44-02 thi-4; lys-1DmatA FGSC 8292

D25 his-5 tol A/a partial diploid This study

R2-11 nic-2 al-1; pan-1 am33 This study

DNA sequence analyses:A 6.9-kb EcoRI tol1-containing con- (2 to 6 map units; Figure 1). A cosmid containing trp-4 struct was subcloned into overlapping fragments suitable for (G4:A9) was identified from a genomic library using the DNA sequencing. DNA sequences were determined for both

trp-4 gene (VollmerandYanofsky1986) as a probe. A strands using the ABI (Mississauga, ON) automated Taq

Dye-chromosome walk was initiated from G4:A9 in both Deoxy Terminator cycle method at the NAPS unit,

Biotechnol-ogy Laboratory, University of British Columbia. Computer directions. Twelve overlapping cosmids spanning a re-sequence analyses for protein and DNA were done using the gion of 350 kb around the trp-4 locus were isolated MacVector/AssemblyLIGN software (International

Biotech-(Figure 1). The physical location of selected cosmids nologies, New Haven, CT) and GCG package available from

was determined using RFLP mapping (Metzenberget the Wisconsin Genetics Computer Group (Devereux et al.

al. 1985) and all mapped to trp-4. 1984).

To determine the orientation of the walk from trp-4, we analyzed progeny that contained crossovers between RESULTS

his-5, tol, and trp-4 from a cross between R5-27 (un-3; his-5 tol trp-4 a; Oak Ridge background) and MV1c-A

Cloning of tol: The tol locus is on the right arm of

LG IV and is flanked by trp-4 (z1 map unit) and his-5 (FGSC 2225; Mauriceville background). Genomic DNA


Figure 2.—The tol locus was bracketed by genetic crossovers and RFLP analy-sis of progeny. (A) Cross-overs between tol and trp-4 were detected in progeny 43-9 and 43-31 (his-51 tol1 trp-4); hybrid RFLP patterns that differed from both pa-rental strains were observed when genomic DNA was probed with X14:C2. (B) Recombination events be-tween his-5 and tol were se-lected for by isolating A/a partial diploid progeny that were his-51, tol, and trp-4. RFLP analysis of genomic DNA from A/a tol trp-4 progeny identified cross-over points in the cross- overlap-ping region of cosmids G13:C8 and X25:D7. When probed with G13:C8, eight progeny (T4, T5, T10, T15, T18, T21, T35, and T37) dis-played the R5-28 (MV LG IV) pattern and one prog-eny (T11) displayed a hy-brid pattern, indicating that the tol locus resided be-tween G13:C8 and X14:C2 in the cosmid walk.

was isolated from His1Trp2progeny and probed with To bracket the tol locus within the cosmid walk, we needed to detect recombination points that were cen-cosmids shown in Figure 1. Recombination points in two

his-51tol1trp-4 progeny (43-9 and 43-31) were identified tromere proximal to tol, i.e., between tol and his-5. A cross was performed between a R5-28 (T(ILIIR) when cosmid X14:C2 was used as a probe (Figure 2A).

These results indicated that cosmids directed toward 39311 ser-3 A; MV LG IV)3 R5-27 (un-3; his-5 tol trp-4 a) (Figure 2B). The R5-28 strain contains an insertional G13:C8 were oriented toward the tol locus, and that the


in-cludes ser-3, un-3, and mat) is inserted into the right tified at position2413, 85 bp away from the proposed transcription start site (Figure 3). Three pairs of short arm of LG II (PerkinsandBarry1977). Progeny were

selected that displayed wild-type growth in medium lack- perfect repeats (CGCCGCCCA, TTTGTTG, and GAGA AGTTCA) were found 59to the proposed CAAT box. ing serine at 308 (selection for A/a tol partial diploid

progeny; Figure 2B) and that contained tryptophan but Translation of the tol ORF identified a 1011-aa poly-peptide (Figure 3) with a calculated Mrof 113,712. TOL

lacked histidine (selection for progeny with

recombina-tion between his-5 and tol). Genomic DNA from 68 A/a has a predicted isoelectric point of 4.67 and is made up of 40% nonpolar and 26% charged residues. The tol trp-4 progeny were screened for RFLP differences

from the R5-27 parent when probed with the cosmids carboxyl-terminal portion of TOL (aa position 837– 1011) is rather hydrophilic, composed of 30% nonpolar centromere proximal to G4:A9. Fifty-nine progeny

con-tained RFLPs that were identical to the R5-27 parent, and 30% charged residues. BLAST (Altschul et al. 1990) and FASTA algorithm (Pearson and Lipman indicating that in these progeny the recombination

point between his-5 and tol lay centromere proximal to 1988) searches did not reveal significant similarity be-tween TOL and any other protein sequence present in the cosmids identified in the walk. In genomic DNA

from eight progeny, an R5-28 pattern was identified protein databases. However, a heptad repeat structure was identified from aa position 177–211 (Figure 4, A when G13:C8 was used as a probe, but an R5-27 pattern

was observed when probed with X25:D7 (Figure 2B). and B), as predicted by the COILS program (Lupaset al. 1991). A coiled-coil is an a-helical bundle that is These data indicated that the recombination point

be-tween his-5 and tol in these progeny occurred bebe-tween thought to wind into a superhelix, with the hydrophobic residues (a and d) forming the hydrophobic packing G13:C8 and X25:D7, and that tol must be centromere

distal to the crossover point. When genomic DNA from interface (for review see Lupas 1996). Coiled-coil do-mains play structural roles in numerous fibrous proteins progeny T11 was probed with G13:C8, a hybrid RFLP

pattern was identified, consistent with the interpretation (PaulingandCorey 1951; Crick1953) and are also a dimerization motif in the leucine-zipper class of tran-that the tol locus resided between G13:C8 and G4:A9.

The Tol1 activity of contiguous cosmids between scription factors (Landschulzet al. 1988). The putative coiled-coil domain in TOL had five heptad repeats and G13:C8 and G4:A9 was assayed by cotransforming each

cosmid with pOKEmat A-1 into R4-71 (his-5 tol trp-4; contained mostly hydrophobic residues in positions a and d with some charged residues in positions b, c, e, pan-2 a) and R4-72 (ad-3B; trp-4 cot-1; pan-2 A)

sphero-plasts. Transformants were selected for both hygro- f, and g. Occasional polar residues in the core (a and d, such as in the case of TOL) favor dimerization over mycin resistance (cosmid marker) and for growth in the

absence of panthothenic acid (pOKEmat A-1 marker). trimer- or tetramerization, because they can still be partly solvated (Harbury et al. 1993; Lumb and Kim Transformants fail to regenerate following the

induc-tion of mating-type-associated incompatibility (Glasset 1995).

A single putative leucine-rich repeat (LRR) was found al. 1990) and therefore a cosmid with Tol1 activity

should exhibit significantly lower transformation fre- in the carboxyl-terminal portion of TOL (position 804– 823; Figure 4A). LRRs, which have been found in quencies when introduced with mat A-1 into tol a

sphero-plasts as compared to A spherosphero-plasts. Only cosmid numerous proteins, are thought to mediate protein-protein interactions (for reviews, seeKobeand Deisen-X25:D7 (plus mat A-1) exhibited a significant reduction

in transformation frequency in a tol a background. Fur- hofer1994;BuchananandGay1996). Each LRR con-tributes an exposed b-sheet that could participate in ther subcloning identified a 6.9-kb EcoRI fragment from

X25:D7 that caused a 20- to 30-fold reduction in trans- strong interactions with other proteins. The LRR in TOL is predicted to form ab-sheet by both the Chou-formation frequencies when introduced into tol a

sphe-roplasts as compared to A sphesphe-roplasts. Fasman (Chou and Fasman 1978) and the Robson-Garnier algorithms (Garnier et al. 1978). Although

Molecular characterization oftol:DNA sequence

de-termination of a 4.2-kb Sal I-Nsi I construct within the LRRs are usually present in tandem arrays, at least three proteins with a single LRR have been reported: Rev 6.9-kb EcoRI fragment revealed an ORF of 3127 bp

inter-rupted by a putative intron of 94 bp. The 59 and 39 (HIV-1 nuclear regulatory protein;Malimet al. 1989), GPIbb (human platelet glycoprotein Ib b subunit; and internal sites of the tol intron fit intron splicing

consensus sequences (Bruchez et al. 1993; Figure 3). Lopezet al. 1988), and GPIX (human platelet glycopro-tein IX; Hickey et al. 1989). These single LRRs are The sequence of a tol cDNA spanning the intron site

was characterized and confirmed the presence of the thought to be functionally important (Malimet al. 1989; Noris et al. 1997).

intron in the genomic tol sequence. DNA sequences

surrounding the tol ATG start codon were in good agree- Functional analysis oftolconstructs:Several tol dele-tion and frameshift constructs were obtained to deter-ment with the consensus for N. crassa (Edelman and

Staben1994). A sequence matching the transcription mine what portions of TOL were important for function (Figure 5). A 11 frameshift mutation at the XhoI site initiation consensus for N. crassa was found at position


iden-Figure 3.—Nucleotide and amino acid sequence of the tol gene of N. crassa. The nucleotide sequence (4.2-kb Sal I-Nsi I fragment) and the predicted TOL protein sequence are shown. The in-tron sequence is italicized and the 59, 39, and inter-nal splicing consensus se-quences are underlined. Se-quences matching the con-sensus for a transcriptional start site and CAAT box are underlined in bold. Short repetitive sequences (three pairs) in the promoter re-gion are underlined. The putative coiled-coil domain and the leucine-rich repeat (LRR) are bracketed with {} and [], respectively. The original tol mutation, E858 (GAA) → Stop (TAA) is underlined in bold and itali-cized. The GenBank acces-sion number for this se-quence is AF085183.

portion also abolished Tol1activity. However, an inter- otic pan-21 tol a (pOKE103, SE5.1, or pOKE-EE6.9) transformants were isolated and subjected to nal deletion of 139 aa in the region before the LRR did

not affect TOL function. N-terminal deletion constructs heterokaryon tests with tol1 A (I-20-26) and tol A (I-20-41) testers. As expected, homokaryotic pan-21 tol indicated that a region between aa position 98 and 531,

which includes the coiled-coil domain, was also essen- a transformants containing only the vector (pOKE103) formed a vigorous heterokaryon with the tol A (I-20-41) tial.

Complementation of the tol mutant and DNA se- strain, but formed an incompatible heterokaryon with

the tol1 A tester (I-20-26). In contrast, the pan-21tol a quence analysis of thetol2allele:The original tol

muta-tion was identified in an A/a partial diploid strain that (pOKE-SE5.1 or pOKE-EE6.9) transformants exhibited typical mating-type-associated incompatibility with both had escaped from inhibited to wild-type growth rates

(Newmeyer 1970). The mutation was subsequently tol A and tol1A testers. These data indicated the original tol mutant phenotype was reverted to wild type (Tol1) mapped to LG IV, near trp-4. To confirm that we cloned

tol, we performed complementation experiments with by the introduction of either of the pOKE-SE5.1 or pOKE-EE6.9 clones. To confirm further that we had the tol2mutant and determined the DNA sequence of

the tol2allele (N83). cloned tol, we determined the DNA sequence of the tol2 mutant allele. The tol2 allele (N83) contained a Two pOKE103-tol1 constructs, Sal I-EcoRI (5.1 kb;

pOKE-SE5.1) and EcoRI-EcoRI (6.9 kb; pOKE-EE6.9; Fig- transversion mutation at position 2666 (G → T) that resulted in the change of a glutamine codon (GAA) ure 5), plus a vector control (pOKE103), were


Figure4.—Structural features identified in TOL. (A) The amino acid sequence of the coiled-coil domain (position 177– 211) in TOL is shown with a schematic heptad position a-g underneath. The leucine-rich repeat (LRR; aa position 804– 823) is shown and the consensus for Arabidopsis thaliana RPS2 LRR (Mindrinoset al. 1994) is depicted underneath. For the LLR, an “X” stands for an arbitrary amino acid while an “a” stands for an aliphatic amino acid. (B) A helical wheel repre-sentation of the coiled-coil domain in TOL. The hydrophobic residues of the heptad repeats are in bold while charged

resi-Figure5.—Functional analysis of tol deletion and frameshift dues are underlined.

constructs. (A) Schematic representation of the 6.9-kb tol DNA sequence including relevant restriction sites. DNA position is given according to Figure 3. (B) Schematic diagram of various

Isolation of tolmutants by RIP mutation:The muta- tol constructs used to examine functional domains of TOL.

Amino acid position is according to Figure 3. A tol construct tion in the original tol2allele (N83) occurred near the

is considered to have TOL activity if it results in a reduction carboxyl-terminus of TOL, and therefore it is possible

in transformation frequency (at least 20-fold) when cotrans-that TOL retains partial function. We therefore at- formed with mat A-1 into tol a spheroplasts, as compared to tempted to obtain additional tol mutants by RIP muta- frequencies when cotransformed into A spheroplasts. *XhoI frameshift contains 50 functional amino acids and terminates tion (Selker1990). RIP is a mechanism in Neurospora

at position 68. that causes duplicated sequences to undergo multiple

G-C to A-T transition mutations during the sexual cycle.

A 2.7-kb EcoRV fragment (cloned into pCB1004) that as RIP strains (to1-43, 83, 95, 106, and tol-contained an internal portion of tol (position 3–2735; 135).

Figure 3) was transformed into strain A2 (ad-3A nic-2 To confirm that the suppressor phenotype in the tol-cyh-1 a; Table 1). A hygromycin-resistant homokaryotic RIP mutants segregated with the tol locus, tol-43 a (as a transformant (4-9) was obtained and crossed with FGSC male) and tol-83 a (as a female) were crossed separately 456 (his-5 A). Forty-nine his-51; ade-3A nic-2 a progeny to his-5 A. Of the 30 His1progeny tested from the two

were analyzed for Tol1 activity in heterokaryon tests. crosses, all but one formed vigorous heterokaryons with Five his-51; ad-3A nic-2 a progeny formed vigorous het- both tol a (I-10-1) and tol A (I-20-41) strains (3.3% recom-erokaryons with the tol A tester (I-20-41), indicating that bination), indicating that the new mutations in tol-43 the tol allele in these strains was not functioning to and tol-83 were closely linked to the his-5 (and hence confer mating-type-associated incompatibility (Figure tol) locus. By Southern blot and RFLP analysis, it was 6A). Heterokaryons between these same five his-51; ad- determined that the tol sequence in the tol-RIP strains 3A nic-2 a progeny and the tol1 A tester (I-20-26) dis- showed restriction site changes (Figure 6B), consistent played typical heterokaryon incompatibility (Figure with the presence of transition mutations that are char-6A), indicating that the tol mutation in these strains was acteristic of sequences that have undergone RIP ( Sel-recessive. The tol-RIP strains were phenotypically normal ker1990).


yon (A1am33); the am33strain forms compatible

hetero-karyons with A strains, but sexual function is not affected (GriffithsandDelange1978).

Following fertilization, opposite mating-type nuclei coexist and divide within a common cytoplasm. There-fore, mating-type-associated incompatibility mediated by tol must be suppressed during sexual reproduction. To determine if tol is transcriptionally repressed dur-ing a cross, thus allowdur-ing the coexistence of opposite mating-type nuclei, we analyzed the presence of the tol cDNA by RT-PCR in mRNA preparations from peri-thecia at 3, 5, 6, and 9 days postfertilization. Although a cDNA for mat A-2 could easily be detected by RT-PCR from perithecial RNA preparations for all time points, a tol cDNA could not be detected at any time point postfertilization (cross; Figure 7 and data not shown).


This study describes the cloning, characterization, and expression analysis of tol, a mediator of mating-type-associated incompatibility in N. crassa. The tol locus is the only molecularly characterized mediator of allelic incompatibility from fungi. Nonallelic suppressors of incompatibility have been characterized in Podospora an-serina and encode proteins thought to be involved in developmental processes and signal transduction ( Lou-bradou et al. 1997;Barreauet al. 1998). In N. crassa, it is not clear how tol, mat a-1, and mat A-1 function to Figure6.—Phenotypic and genotypic analysis of tol-RIP

mu-trigger incompatibility during vegetative growth. One tants. (A) The top three plates are controls showing growth

characteristics of compatible (tol A1tol a) and incompatible hypothesis is that, in an A 1 a heterokaryon or A/a (tol1A1tol a and tol1A1tol1a) heterokaryons. The bottom partial diploid, MAT a-1 and MAT A-1 form a hetero-plates show the phenotype of heterokaryon with tol-43. The

dimer and this complex acts as a transcription regulator tol-43 strain was compatible with a tol A strain, but incompatible

to activate or repress the expression of tol (Glassand with a tol1 A strain. Strains I-10-41 (tol A), I-20-26 (tol1A),

Staben1990), which would then trigger incompatibil-I-10-1 (tol a), I-1-51 (tol1a), and R5-52 (tol-43 a) (Table 1)

were used in heterokaryon tests. (B) RFLP analysis of tol se- ity. Our studies show that the expression of tol occurs quences in the tol-RIP strains. Genomic DNA from parental in an A, a, and even in aDmat strain. These data indicate strains (his-5 and transformant 4-9) plus the transformation

that MAT A-1 and MAT a-1 are not required for either recipient strain (A2; Table 1) and each tol-RIP strain (tol-43,

repression or activation of tol, although we cannot rule tol-83, tol-95, tol-106, and tol-135) was digested with MboI (M)

out the possibility that the mating-type polypeptides may and Sau3AI (S) and probed with a 3.5-kb fragment containing

tol. The presence of altered restriction patterns in the RIP modulate tol expression levels. The expression of tol was progeny as compared to their parental controls is indicative also detected in an A/a tol partial diploid and an A1 of GC→AT transition mutations that occur during the RIP

a incompatible heterokaryon, indicating that if a MAT process (Selker1990).

A-1/MAT a-1 heterodimer is formed, it does not materi-ally affect the expression of tol.

On the basis of the data presented in this article, a used to amplify tol mRNA from vegetatively growing

cultures. Figure 7 shows that a tol cDNA could be de- second more plausible possibility for the mechanism of mating-type-associated incompatibility can be formu-tected from mRNA from A, a, and a mating-type deletion

strain grown in vegetative and crossing media (V and lated. In our current hypothesis, TOL physically inter-acts with the MAT A-1/MAT a-1 complex or their down-C; Figure 7). These data indicated that neither

mating-type constitution nor growth conditions materially af- stream products to trigger incompatibility. The 1011-aa TOL protein possesses a putative coiled-coil domain and fected the expression of tol. A tol cDNA could also be

detected in an A/a tol partial diploid strain, indicating a LRR, both of which are thought to mediate protein-protein interactions. Putative LRRs are found in both that the presence of both mat A and mat a in the same

nucleus did not significantly alter activation or repres- MAT A-1 and MAT a-1 (P. K. T. Shiu, unpublished results), and alteration of these motifs affects the capac-sion of tol. Exprescapac-sion of tol was also detectable in both


Figure7.—Expression of tol as analyzed by RT-PCR. Primers spanning the in-tron (tol 13 and tol 11) were used to amplify tol cDNA from different mRNA prep-arations. The size of the ge-nomic tol product is 504 bp with these primers, while the tol cDNA product is 410 bp. Strains used are as fol-low: 74-OR23-1VA (A), OR8-1a (a), RLM44-02 (DmatA), D25 (A/a tol), I-20-261 I-1-51 (A1a), and I-20-261 R2-11 (A1am33). mRNA was

iso-lated from the above cul-tures grown either in vegeta-tive medium (Vogel 1964; V) or crossing medium (Westergaardand Mitch-ell1947; C) or from perithe-cia (cross). A tol cDNA could be detected in mRNA from all strains, with the exception of peritheperithe-cial mRNA. The quality of the perithecial mRNA samples was checked by the amplification of a mat A-2 mRNA (using primers rI.1 and 2423-2406;Ferreira et al. 1996); mat A-2 is expressed at low levels throughout the sexual cycle (data not shown). DNA size markers (1-kb ladder) are given on the left.

A 4-aa deletion in the carboxyl terminus of MAT a-1 a vigorous heterokaryon. However, when mat A and mat a from N. sitophila were introgressed into N. crassa, abolished incompatibility but not DNA binding or

mat-ing activity (Philley and Staben 1994). These data mating-type-associated incompatibility was observed (Perkins 1977). In the pseudohomothallic species N. suggest that for MAT a-1, mating (which probably

in-volves transcriptional activation) and vegetative incom- tetrasperma, opposite-mating-type nuclei normally re-side in a common cytoplasm. Similar to the results ob-patibility are mediated by biochemically distinct

mecha-nisms. served with N. sitophila, mat A and mat a from N.

tetra-sperma displayed mating-type incompatibility when in-Some het genes have cellular functions other than to

limit heterokaryosis (for review, see Be´gueret et al. trogressed into N. crassa (Metzenberg andAhlgren 1973). The introgression of tol1from N. crassa into N. 1994). The original tol mutant and our tol -RIP strains

exhibit normal vegetative growth and sexual reproduc- tetrasperma induced mating-type-associated incompati-bility and disrupted the pseudohomothallic nature of tion. However, the tol mutation has been shown to

sup-press the phenotype of an fmf-1 mutant (Johnson the species (Jacobson 1992). All of these data argue that tol is the major mediator of mating-type-associated 1979), which is sterile as both a male and a female in

crosses to a wild-type partner. Fertility is restored when incompatibility. The evolutionary history and selection for or against mating-type-associated incompatibility in an fmf-1 tol strain is crossed to an fmf-11tol strain. Our

expression analysis of tol is consistent with the hypothesis the different species within the genus Neurospora re-main an enigma. The molecular characterization of tol that tol activity is suppressed during sexual development

to allow the coexistence of opposite mating-type nuclei from N. crassa and related species will provide the neces-sary tools to address these questions.

in the ascogenous hyphae. It is possible that FMF-1

regu-lates tol either directly or indirectly, and thus may play Both allelic and nonallelic incompatibility systems have been described for a number of filamentous fungi, a role in the repression of tol transcription.

Presum-ably, the misexpression of tol during the sexual cycle and it is believed that heterokaryon (or vegetative, so-matic, or heterogenic) incompatibility is a universal would result in mating-type-associated incompatibility

and block further sexual development. The presence phenomenon among filamentous ascomycetes and basi-diomycetes (Glass and Kuldau 1992; Leslie 1993; of short repetitive sequences in the promoter of tol may

provide putative binding sites for such regulators. Be´gueretet al. 1994;EsserandBlaich1994;Worrall 1997). It has been proposed that vegetative incompati-Some species within the genus Neurospora do not

exhibit mating-type-associated incompatibility. This fact bility may protect an individual from the transfer of deleterious cytoplasmic factors (Caten1972). Cytoplas-is presumably due to the presence of tol mutations within

these species that suppress mating-type-associated in- mic transfer of hypovirulence-associated dsRNA virus in Cryphonectria parasitica and detrimental KalDNA plas-compatibility. In the heterothallic species N. sitophila,


Beadle, G. W.,andV. L. Coonradt,1944 Heterokaryosis in Neuro-(Anagnostakis1982;Debetset al. 1994). In black

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10:441–446. Vegetative incompatibility mediated by mating type

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Carroll, A. M., J. A. SweigardandB. Valent, 1994 Improved N. crassa; N. crassa populations are highly polymorphic

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Chou, P. Y.,andG. D. Fasman,1978 Prediction of the secondary morphic in populations in a heterothallic species like

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Esser, K.,andR. Blaich,1994 Heterogenic incompatibility in fungi, A–1, mat a-1, and tol, will provide tools to analyze the

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Figure 5.—Functional analysis ofconstructs. (A) Schematic representation of the 6.9-kbsequence including relevant restriction sites


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