HET-E and HET-D Belong to a New Subfamily of WD40 Proteins Involved in Vegetative Incompatibility Specificity in the Fungus Podospora anserina

Copyright 2002 by the Genetics Society of America

HET-E and HET-D Belong to a New Subfamily of WD40 Proteins Involved inVegetative Incompatibility Specificity in the Fungus Podospora anserina

Eric Espagne,1 Pascale Balhadere,2 Marie-Louise Penin, Christian Barreau and Beatrice Turcq3

Institut de Biochimie et de Genetique Cellulaires, CNRS UMR 5095, 33077 Bordeaux, France

Manuscript received November 5, 2001Accepted for publication February 8, 2002

ABSTRACTVegetative incompatibility, which is very common in filamentous fungi, prevents a viable heterokaryotic

cell from being formed by the fusion of filaments from two different wild-type strains. Such incompatibilityis always the consequence of at least one genetic difference in specific genes (het genes). In Podosporaanserina, alleles of the het-e and het-d loci control heterokaryon viability through genetic interactions withalleles of the unlinked het-c locus. The het-d2Y gene was isolated and shown to have strong similarity withthe previously described het-e1A gene. Like the HET-E protein, the HET-D putative protein displayed aGTP-binding domain and seemed to require a minimal number of 11 WD40 repeats to be active inincompatibility. Apart from incompatibility specificity, no other function could be identified by disruptingthe het-d gene. Sequence comparison of different het-e alleles suggested that het-e specificity is determinedby the sequence of the WD40 repeat domain. In particular, the amino acids present on the upper faceof the predicted �-propeller structure defined by this domain may confer the incompatible interactionspecificity.

IN filamentous fungi, anastomoses between hyphal compatibility results from the coexpression of two antag-filaments occur frequently and produce heterokary- onistic alleles from a single het locus, this is called an

otic cells in which two different genomes coexist. Heter- allelic incompatibility system; if, conversely, incompati-okaryotic cell viability is controlled by specific het loci bility results from the coexpression of two antagonisticinvolved in vegetative or heterokaryon incompatibility alleles from two separate het loci, this is known as a(Esser and Blaich 1973; Glass and Kuldau 1992; nonallelic incompatibility system (Bernet 1965).Begueret et al. 1994; Loubradou and Turcq 2000; Characterization of het genes is the first step towardSaupe 2000). In Podospora anserina, a single genetic dif- understanding the phenomenon of vegetative incom-ference between the two genomes at one het locus is patibility. Several genes involved in vegetative incompat-sufficient to impair the viability of the resulting hetero- ibility have been characterized in fungi. In N. crassa, thekaryotic cell (Rizet 1952; Bernet 1965). A lytic process mating-type alleles (mat a-1 and mat A-1), which alsorapidly destroys the heterokaryotic cell (Beisson-Sche- control vegetative incompatibility, have been studiedcroun 1962). Accumulation of dead cells between two extensively (Glass et al. 1988, 1990; Staben and Yanof-incompatible strains leads to formation of an abnormal sky 1990): the het-C encoded protein displays a glycine-contact zone called “barrage” (Rizet 1952). rich domain (Saupe et al. 1996), the het-6 gene product

The number of het loci is generally high: 11 in Neurospora presents sequence similarity with P. anserina HET-Ecrassa (Perkins et al. 1982), 9 in P. anserina (Bernet 1967), (Smith et al. 2000), and the un-24 gene encodes the8 in Aspergillus nidulans (Grindle 1963) and 6 or 7 in large subunit of type I ribonucleotide reductase (SmithCryphonectria parasitica (Anagnostakis 1977; Cortesi et al. 2000). In P. anserina, the allelic incompatibilityand Milgroom 1998). In P. anserina, two different types het-s gene encodes a prion-like protein (Turcq et al.of incompatibility systems have been described. If in- 1990; Coustou et al. 1997). The het-c gene involved in

both nonallelic systems het-c/het-e and het-c/het-d encodesa protein similar to the glycolipid transfer protein, andinactivation of this gene leads to abnormal ascosporeSequence data from this article have been deposited with the

EMBL/GenBank Data Libraries under accession nos. AF323585, formation (Saupe et al. 1994). The het-e gene encodesAF323582, and AF323583. a protein that displays a GTP-binding site and a WD40

1Present address: Institut de Recherche sur la biologie de l’insecte, repeat domain. Previous studies have demonstrated thatUPRESA 6035, Universite F. Rabelais, 37200 Tours, France.the P. anserina het-e allele reactivity in incompatibility is2Present address: School of Biological Sciences, University of Exeter,

Exeter EX4 4QG, England. dependent on both GTP binding by the P-loop domain3Corresponding author: Laboratoire de Biologie et Genomique de and a minimal number of 10 WD40 repeats (Saupe et al.

Podospora, Institut de Biochimie et de Genetique Cellulaires, CNRS 1995; Espagne et al. 1997). Investigation of differentUMR 5095, 1 rue Camille Saint-Saens, 33077 Bordeaux Cedex, France.E-mail: [email protected] het-e alleles, which were reactive in incompatibility,

Genetics 161: 71–81 (May 2002)

72 E. Espagne et al.

analyses have been described (Rizet and Engelman 1949;showed that the number of WD40 repeats does notEsser 1974). Vegetative incompatibility can be determined bycorrelate with allele specificity.strain confrontation on solid corn-meal agar. Incompatibility

The WD40 repeat, first identified in the �-subunit of results in the formation of a barrage, i.e., a dense unpigmenteda G-protein, is a degenerate sequence repeat of �40–43 line in the contact region between two mycelia of antagonistic

strains (Rizet 1952).amino acids in length (Fong et al. 1986). HeterotrimericThe two nonallelic incompatibility systems het-c/het-e andG-protein structure has revealed that the seven WD40

het-c/het-d are characterized by multiple alleles present at therepeats of the G� subunit are organized in a circularhet-c, het-e, and het-d loci in wild-type isolates. All strains used

structure of seven �-sheets, forming the blades of a in this study were isogenic to wild-type isolate s (Rizet 1952)�-propeller structure around a central pore (Lam- but with different alleles at het-c, het-e, and het-d loci. Among

17 wild-type strains studied by Bernet (1967), four classes ofbright et al. 1996; Sondek et al. 1996). Upper, lower,het-c alleles, four classes of het-e alleles, and three classes ofand circumferential faces are potential binding surfaceshet-d alleles have been described. An allele is designated by(Smith et al. 1999). Each �-sheet is composed of fourthe class number and the name of the wild-type isolate. For

antiparallel strands. The same structure has been de- example, the allele het-d2 of the Y strain is denoted het-d2 Y. Ifscribed for the C-terminal domain of TUP1, which also a strain contains a neutral allele at any of the three loci, this

locus is not mentioned in the strain designation.displays seven WD40 repeats (Sprague et al. 2000). InDNA analysis: Standard techniques were used for DNA clon-addition, the biochemical properties of four other

ing, restriction enzyme digestion, Southern blot analysis, andWD40 repeat proteins, with repeats ranging from fiveDNA sequencing (Sambrook et al. 1989). Genomic DNA was

to seven, suggest that all WD40 repeats fold into a similar prepared with the rapid petri-dish-grown mycelia methodtertiary structure (Garcia-Higuera et al. 1996). The (Lecellier and Silar 1994). For the library construction,

genomic DNA was prepared as described ( Javerzat et al.�-propeller structure was also described in non-WD40-1993). For het-d sequencing, exonuclease III sequential dele-repeat proteins (Smith et al. 1999). The highest-knowntions were performed using an ExoIII/mung bean nucleasenumber of blades in a propeller structure is eight anddeletion kit (Stratagene, La Jolla, CA) as recommended by

the highest-known number of WD40 repeats is 16. For the supplier. To determine the number of WD40 repeats ina protein containing more than eight WD40 repeats, it different het-d alleles, genomic DNA was digested by BamHI

and SphI and probed with the fragment SphI-XhoI [nucleotideremains unclear whether the protein forms two small(nt) 2578–2925] located upstream from the repeat region.propellers or a large one (Smith et al. 1999). The poten-

DNA library construction: The DNA library was constructedtial role of HET-E WD40 repeats in a biological functioninto the pMOcosX cosmid (Orbach 1994) using the XL kit

and the mechanism of regulating vegetative incompati- (Stratagene). Partially SalI-restricted DNA fragments from thebility both remain unclear. Inactivation of the het-e gene P. anserina Y strain were size selected by pulsed field gel elec-

trophoresis performed in 0.5� TBE at 9� using a contour-does not lead to any particular phenotype other thanclamped homogeneous electric field apparatus (LKB, Pis-the incompatibility phenotype.cataway, NJ) on 1% low-melting-point agarose gel (SeaPlaqueSouthern blot analyses on genomic DNA have suggestedagarose, FMC, Rockland, ME). Samples migrated for 16 hr in

the existence of a sequence similar to het-e (Espagne et an electric field of 180 V with a 5-sec switching time. Gelsal. 1997). It was possible that the het-d gene, the other were then stained for 1 hr in a 0.2-�g/ml ethidium bromide

solution and washed for 30 min in water. The DNA was visual-het-c antagonist, could be a functional homolog of het-e.ized by UV fluorescence. �DNA oligomers (between 48.5 andTo increase our understanding of het genes in vegetative485 kbp) were used as size markers. DNA fragments betweenincompatibility, we undertook het-d characterization. As25 and 50 kbp were isolated from the gel and agarose elimi-

for HET-E, domains characteristic of both �- and �-sub- nated by GELase treatment (GELase, Epicentre, Madison,units of a heterotrimeric G-protein (Espagne et al. 1997), WI). The pMOcosX vector was restricted by XbaI to generate

vector arms with cos extremities, treated with alkaline phos-were also found in HET-D putative protein. All the het-dphatase, and then restricted with XhoI. Ligation was performedalleles reactive in incompatibility had a minimal numberfor 16 hr at 26� in 10 �l of ligation buffer using �2 �g ofof 11 full-length WD40 repeats.purified genomic fragments, 4 �g of vector arms, and 5 units

To identify the domains potentially involved in the of T4 DNA ligase (Life Technologies). The ligation mix wasvegetative incompatibility specificity, we compared the then used for in vitro packaging (Gigapack II Gold kit, Stra-

tagene) and transfection into Escherichia coli DH5�, as de-amino acid sequence of different alleles of het-e ratherscribed by the manufacturer.than of het-d alleles, because first, four different allele

Protoplasts were prepared and transformed as previouslyspecificities have been described for het-e alleles anddescribed (Berges and Barreau 1989). The pMOcosX vector

only three for het-d alleles, and second, because only contained the bacterial hygromycin resistance gene hph as amutant alleles reactive or nonreactive in incompatibility selectable marker (Orbach 1994), and transformants were

screened on hygromycin B at 100 �g/ml.are available for het-e. The results showed a crucial roleof the WD40 repeat sequence on the vegetative incom-patibility specificity.

RESULTS

Cloning of the incompatible het-d2Y gene by pheno-MATERIALS AND METHODS typic expression: To clone the het-d2Y gene, a genomic

cosmid library from the Y wild-type strain was dividedStrains and growth conditions: P. anserina is a heterothallicascomycete. Its life cycle and general methods for genetic into 16 pools of 192 cosmids. DNA prepared from each

73Vegetative Incompatibility in Fungi

pool was used to transform a recipient strain containing reaction: they had gained the het-d2Y phenotype. Thecosmid conferring this phenotype was isolated from thea het-c allele null in incompatibility. Hygromycin-resis-

tant transformants were tested for incompatibility with cosmid pool after three rounds of sib-selection (Akinsthe strain containing the het-c4M allele antagonistic to and Lambowitz 1985). The functional het-d2Y gene wasthe het-d2Y allele by screening for barrage formation then subcloned on a 5.2-kbp SalI-XbaI fragment, whichwith this tester strain. Three strains, arising from trans- was then sequenced.formation with the same DNA pool, showed a barrage The het-d2Y gene encodes a protein showing high simi-

larity with HET-E1A protein: Sequence analysis revealedthat the 5.2-kbp fragment contained an open readingframe (ORF) of 4187 bp interrupted by a putative intronof 56 bp, according to consensus sequences for filamen-tous fungi splicing sites (Ballance 1991; Bruchez etal. 1993). This putative intron splits the open readingframe into two exons, each of them encoding a distinct,characteristic domain. Such a structure has already beenreported for the het-e1A gene (Saupe et al. 1995). Al-though these two genes show 66.5% nucleotide identity,some minor differences were observed. The intron is lo-cated at a position corresponding to the 751st amino acidfor the predicted het-d2Y polypeptide and to the 761stamino acid for the protein encoded by het-e1A gene. Thehet-dY intron is 56 bp, whereas the het-e1A intron is only49 bp; in addition, the splicing site sequences are differ-ent for the two introns (data not shown).

The het-d2Y gene encodes a 1376-amino-acid putativepolypeptide with a predicted molecular mass of 152 kD,displaying high similarity (53% identity, 71% similarity)with the HET-E1A protein (Saupe et al. 1995; Espagneet al. 1997). Sequence analyses show that a GTP-bindingsite (Saraste et al. 1990; Bourne et al. 1991) and aWD40 domain (Van der Voorn and Ploegh 1992) arefound in N-terminal and C-terminal regions, respec-tively (Figure 1). This structure is identical to that ofthe HET-E1A protein (Saupe et al. 1995). Positions of theGTP-binding-site P-loop and G2, G-3, and G-4 sequencesare highly conserved between HET-D2Y and HET-E1A,but only the P-loop sequences are identical (Figure 1B).The HET-D2Y WD40 domain is very similar to the HET-E1A WD40 domain, although HET-D2Y and HET-E1A donot share any identical repeats. Equally, the HET-D2Y

domain is longer, as it contains 12 WD40 repeats (11full-length repeats and the first 30 amino acids of a 12

Figure 1.—Amino acid sequence of HET-D2Y. (A) Sche-matic representation of HET-D2Y polypeptide. (B) Alignmentof HET-D2Y (D, top line: accession no. AF323585) and HET-E1A (E, bottom line: accession no. L28125) polypeptides. TheHET-E1A sequence corresponds to the revised sequence. Peri-ods indicate identity and dashes indicate gaps. The large arrowindicates position of the intron for each sequence. The threesequences described by Smith et al. (2000) defining the threeblocks contained in the conserved domain are in white upper-case letters inside dark boxes. Regions of the GTP-bindingdomain conserved among G-proteins (P-loop, G2, G3, andG4) are underlined (Bourne et al. 1991). Two G2 sequencesare possible for HET-E1A polypeptide (Saupe et al. 1995). Theshaded boxes in the C terminus indicate the WD40 repeats.


Figure 2.—Sequence of the WD40repeats of the HET-D2Y polypeptide.(A) Alignment of the WD40 repeatsof the HET-D2Y protein. The posi-tions that are conserved are lightlyshaded. Uppercase letters in white in-dicate the positions that differ fromthe consensus sequence. They are in-dicated by a gray background (poly-morphic position) or by a black back-ground (highly polymorphic). Spaceshave been introduced to separate thedifferent structural blocks. (B) Con-sensus sequence for the repeats isgiven with the predicted secondarystructure of a repeat schematizedabove (___, a �-strand; $$$, a loop;???, a turn). The letters d, a, b, andc indicate the �-strands d, a, b, andc, respectively. The numbers indicatethe amino acid positions in a repeat.Variable positions in uppercase let-ters are indicated by a gray back-ground (polymorphic) or by a blackbackground (highly polymorphic).Every possible amino acid found atthese positions is reported below, ar-ranged in order of frequency.

repeat) instead of 10 for HET-E1A (Figure 2A). Out of HET-E1A. In fact, only the 242 N-terminal amino acidsof this 605-amino-acid-long polypeptide show similari-the 42 amino acids of a repeat, 31 are conserved within

the 11 full-length repeats. Moreover, some repeats (re- ties to HET-D2Y (53 and 70% of identity and similarity,respectively) and to HET-E1A N-terminal. These identi-peats 3 and 7 and repeats 5 and 10) are identical (Figure

2A). The repeats differ from each other by, at the most, ties are restricted to the blocks corresponding to con-served regions previously described in P. anserina HET-8 amino acids out of 42. Where any two random repeats

are compared, sequence identity is very strong (�80%). E, in N. crassa HET-6, and in N. crassa TOL proteinsinvolved in vegetative incompatibility (Smith et al.The same situation was previously reported for HET-

E1A protein (Saupe et al. 1995). These findings contrast 2000). This conserved region of �150 amino acids dis-plays three blocks of 17, 36, and 10 amino acids, respec-with the situation for other known WD40 proteins,

where identity between any two repeats never exceeds tively. These blocks, believed to represent an incompati-bility domain, are highly conserved between the three20–30% (Saupe et al. 1995). The HET-D2Y WD40 repeat

consensus sequence and its predicted secondary struc- HET-D2Y, HET-E1A, and Q9P654 polypeptides (�50, 33,and 80% similarity, respectively; Figure 3). Outside thisture are reported in Figure 2B. In the first 32 N-terminal

amino acids of the repeats, 4 of the 11 polymorphic region, both HET-D and HET-E polypeptides are com-pletely unrelated to N. crassa incompatibility polypeptides.positions located there are highly polymorphic, with

3–5 different amino acids being observed for a given The number of WD40 repeats in different wild-typehet-d alleles: Our previous results showed that HET-Eposition (Figure 2, A and B). One highly polymorphic

position (amino acid 11) is in the loop between the d reactivity in incompatibility depends on two functionalelements: a WD40 domain with at least 10 repeats and�-strand and the a �-strand; another (amino acid 13)

is in first position on the a �-strand; the remaining a functional GTP-binding domain (Saupe et al. 1995;Espagne et al. 1997). Different wild-type het-d allelestwo positions (amino acids 29 and 31) are in the turn

between the b �-strand and the c �-strand. were then examined to determine the number of WD40repeats by estimating the size of the region encom-A BLAST search in databanks with the N-terminal part

of the HET-D polypeptide identified only two polypeptides passing the repeats (Table 1). Two classes of wild-typehet-d alleles were analyzed: those reactive in incompati-homologous to HET-D2Y. The first polypeptide is HET-

E1A; the second is an open reading frame translation prod- bility (het-d1A, het-d2 F, and het-d2Y) and those nonreactivein incompatibility (het-d3 alleles from B, D, E, H, M, s,uct, Q9P654, issued from the German Neurospora ge-

nome-sequencing project (accession no. AL356324). This U, V, W, X, and Z strains). Southern experiments re-vealed the presence of a 2100-bp fragment for the threeQ9P654 predicted polypeptide is annotated as related

to �-transducin-like protein because of its similarity to active het-d alleles. All three polypeptides encoded by


Figure 3.—Alignment of conserved blocks of HET-D2Y (accession no. AF323585) and HET-E1A (accession no. L28125) predictedproteins from P. anserina and Q9P564 ORF (accession no. AL356324) resembling HET-E1A, HET-6OR (accession no. AF206700),HET-6PA (accession no. AF208542), and TOL (accession no. AF085183) predicted proteins from N. crassa. HET-D2Y, HET-E1A,and Q9P564 sequences were aligned using Clustal with default setting and conserved block aligned to the previously describedregion (Smith et al. 2000). Amino acids present in one of the two P. anserina polypeptides and conserved in the other predictedproteins are indicated by shading. A star indicates that the amino acids at this position are identical in all sequences, a colonrepresents a conservative substitution, and a period a semiconservative substitution. The consensus sequence is called “cons.”Uppercase letters show that the amino acid at this position is identical in all sequences and lowercase letters show that the aminoacid at this position is conserved in at least half of the six sequences.

het-d1A, het-d2F, and het-d2Y active alleles display 12 WD40 tions were found (Table 2). Two of these polymorphicpositions are specific to the null het-e4 s allele: one leadsrepeats (11 full-length and 1 truncated repeat). For

the het-d3 neutral alleles, the size of the hybridizing to an insertion of three amino acids after the aminoacid 1285 in the predicted polypeptide; the other is afragments ranged from 1340 bp (het-d3W allele) to 2225

bp (het-d3 s allele), corresponding to an estimated num- single nucleotide change that modifies the amino acid482 (Pro482 → Ser482). What distinguishes the het-e4s alleleber of 6 and 13 WD40 repeats, respectively (Table 1). All

het-d alleles containing �11 WD40 repeats are neutral from all the sequenced het-e alleles is that it is not reactivein incompatibility. Although the inactivity is probablyalleles. As observed for het-e alleles (Espagne et al. 1997),

a minimum number of repeats seems to be necessary due to the low number of repeats (three repeats), thesetwo positions could be important for reactivity in incom-for an incompatibility reaction; this number is 10 for

HET-E1A and may be 11 for HET-D2Y. However, the patibility. The five other polymorphic positions concernthe het-e1A allele compared to the het-e2C and het-e4 s al-number of repeats does not seem to be the only determi-

nant of activity in incompatibility, since five het-d3 alleles leles. Two positions correspond to synonymous substitu-tions at amino acids 543 and 691. The three other posi-also contain 11 repeats and are neutral. Moreover, the

three alleles reactive in incompatibility encode a poly- tions are nonsynonymous substitutions that modifyamino acids 693, 1309, and 1342 (Table 2). These threepeptide with the same predicted number of repeats

but with a different incompatibility spectrum; thus the amino acid modifications could be responsible for thedifferences between the het-e1 and het-e2 phenotypes.number of repeats is not specific for a given incompati-

ble interaction. To test this hypothesis, other different wild-type het-e allelesthat are reactive in incompatibility (het-e1H, het-e1M, andSequence differences and het-e allele specificity: Allele

specificity was analyzed on het-e rather than on het-d since het-e3F) have been sequenced at these polymorphic posi-tions (Table 2). Amino acids present at positions 693,the number of wild-type het-e alleles reactive in incompat-

ibility is higher. Moreover, only mutant alleles still reac- 1309, and 1342 are identical in het-e1M, het-e2C, and het-e3F

alleles. Since these three alleles do not display the sametive in incompatibility are available for het-e. To deter-mine which domains of the HET-E polypeptide are incompatibility spectrum, these three polymorphic posi-

tions are not responsible for het-e allele specificity. Thesespecific to the incompatibility spectrum, four het-e geneshave been sequenced. One, het-e2C, is reactive in incompati- polymorphic positions lie in the region that shows less

conservation between HET-E1A and HET-D2Y polypep-bility. Another, also reactive in incompatibility, het-e1A, wasresequenced and five differences with the sequence pre- tides. Altogether, it appears that amino acid sequence

outside of the WD40 domain is not responsible for theviously published by Saupe et al. (1995) were found.Only three of these differences (Ala851 → Pro, Arg895- specificity of the incompatibility spectrum.

The incompatibility specificity should be due to differ-Glu → Gly-Gln, and Gly1010 → Asn) modify the proteinsequence in the WD40 domain. The third, het-e4s, is null ence in the WD40 domain. Saupe et al. (1995) and

Espagne et al. (1997) have already established that thein incompatibility. The fourth, het-e2C-4, is a het-e2C mutantallele still reactive in incompatibility. This mutant allele, number of WD40 repeats was not correlated with the

incompatibility specificity. So, the difference in the het-which was obtained by UV mutagenesis and previouslydescribed by Espagne et al. (1997), has lost incompati- e1 and het-e2 incompatibility spectrum should result

from difference in the amino acid sequence of thebility with the het-c3 allele but has retained incompatibil-ity with het-c1 and het-c4 antagonistic alleles. It should be WD40 domain. Wild-type het-e1A and het-e2C and the mu-

tant het-e2C-4 WD40 domains were sequenced (Figurenoted that positions and sequences of the intron wereidentical in all four alleles. 4). Analysis of the sequences confirmed our previous

observations that the repeat number is 10 for theseOutside the WD40 domain, seven polymorphic posi-


TABLE 1

Result of Southern blot analyses of different strains containing wild-type het-d alleles

Interaction withSize (bp) of Repeat

het-d alleles het-c1 het-c2 het-c3 het-c4 the fragment no.

het-d1 A 2100 12het-d2 F 2100 12het-d2 Y 2100 12het-d3 B 2100 12het-d3 D 2100 12het-d3 E 1700 9het-D3 H 1550 8het-d3 M 2100 12het-d3 s 2225 13het-d3 U 1950 11het-d3 V 2100 12het-d3 W 1340 6het-d3 X 1825 10het-d3 Z 2100 12

The phenotype in incompatibility of an het-d allele is determined by its incompatible interaction with thedifferent het-c alleles ( and indicate incompatible and compatible combinations, respectively). GenomicDNA was digested with BamHI and Sph I and probed with the fragment Sph I-XhoI (nt 2578–2925) locatedupstream of the repeat domain. The number of WD40 repeats was deduced by comparing the length of thedetected BamHI-Sph I fragment with the length of the het-d2 Y WD40 fragment, which contains 12 repeats (11full-length repeats and a truncated one).

alleles (Saupe et al. 1995; Espagne et al. 1997). Only between b and c �-strands (amino acids 29 and 31).These polymorphic sites between the het-e1A and het-e2Cthree repeats (1, 2, and 5) display the same sequence

for the encoded polypeptides (Figure 4). A total of 26 alleles have already been reported (Saupe et al. 1995) asvariable positions between the different HET-E1A WD40polymorphic positions were found between the HET-

E1A and HET-E2C WD40 domains (Figure 4). There are repeats (Figure 4A). These positions correspond to thehighly polymorphic positions also observed between thethree positions in the turn following the c �-strand

(amino acids 38 and 39) and one (amino acid 1) in the d different WD40 repeats of the HET-D2Y (Figure 2A). Itcan be observed that highly polymorphic regions are�-strand (Figure 4). The 22 other changes are located

exclusively within three specific regions of the WD40 located at corresponding positions in the same struc-tural element for the WD40 repeats of the HET-E1A andrepeats. Six polymorphic positions are found in a region

corresponding to the loop between the d and a �-strands HET-D2Y polypeptides, suggesting that the assortment ofamino acids present at these critical positions is impor-(amino acids 10 and 11). Seven are located at the first

position of the a �-strand (amino acid 13) and one in tant to determine the specificity (Figures 2A and 4A).Amino acid sequence comparison between HET-E2C-the a �-strand (amino acid 16). Eight occur in the turn

TABLE 2

Polymorphism outside the WD40 domain of different wild-type and mutant het-e alleles

Interaction with Polymorphic positions

het-e alleles het-c1 het-c2 het-c3 het-c4 482 543 691 693 1285 1309 1342

het-e1 A (10) Pro Arg Leu Tyr His Serhet-e1 H (10) het-e1 M (12) Pro Arg Leu Met Tyr Cyshet-e2 C (10) het-e3 F (12) het-e4 S (3) Ser Arg Leu Met Trp-Leu-Pro Tyr Cyshet-e2 C-4 (10) Pro Arg Leu Met Tyr Cys

Numbers of WD40 repeats estimated previously either by Saupe et al. (1995) or Espagne et al. (1997)are indicated in boldface type in parentheses. and indicate incompatible and compatible interactions,respectively. Polymorphic position numbers correspond to HET-E1A sequence. For each allele, amino acidspresent at the polymorphic positions are indicated.


Figure 4.—Amino acidsequence comparison of theWD40 repeat domain of theHET-E1A (A in sequence),HET-E2C (C in sequence),and the mutant HET-E2C-4(C4 in sequence) polypep-tides. (A) Consensus se-quence for HET-E1A repeatsbased on a frequency of atleast 0.4 for a conservedamino acid (aa) at each po-sition. The number indi-cates the aa position in a re-peat. Polymorphic positionsand highly polymorphic po-sitions are indicated by grayand black backgrounds, re-spectively. Above the con-sensus sequence, the pre-dicted secondary structureis schematized (__, for a �-strand; $$$, for a loop; ???,for a turn). The letters d, a,b, and c indicate the �-strands d, a, b, and c, respec-tively. (B) Alignment of wild-type and mutant HET-EWD40 repeats. Amino acidpositions in the sequence areindicated at the right end ofeach repetition. For HET-E2C

and HET-E2C-4, only aminoacids that differ from thoseof HET-E1A are given. Poly-morphic positions betweenHET-E1A and HET-E2C areindicated by shading anddifferences between HET-E2C and HET-E2C-4 are indi-cated by a black background.

4 and HET-E2C sequences shows five differences in the 6-kbp SmaI-XbaI fragment, was disrupted (Figure 5A).The two SalI fragments (1000 and 1547 bp) encom-WD40 repeated domain. These changes are located

within the sixth and seventh repeats (Figure 4B). Strik- passing the N-terminal conserved region and the GTP-binding domain were replaced by the ura5 gene (Turcqingly, these five mutations modify amino acids at posi-

tions corresponding to the highly polymorphic posi- and Begueret 1987). The resulting plasmid, pS�D, wasused to transform a recipient strain carrying the het-d2Ftions in HET-E2C and HET-E1A repeats. Amazingly, four

of these mutations change the amino acid present in allele, which is reactive in incompatibility, as well as theura 5-6 mutation. To screen transformed strains thatthe HET-E2C-4 sequence into an amino acid identical

to the one present in the HET-E1A sequence (positions have lost the het-d2F specificity, 2500 prototrophic trans-formants were confronted with a tester strain containing1055, 1073, 1095, and 1097). For the fifth mutation

(position 1071), an amino acid, different from the one the antagonistic het-c4M allele. Four transformants dis-playing a neutral incompatibility phenotype were ob-in HET-E1A and in HET-E2C, is present. The het-e2C-4

gene is a het-e2C mutant allele that has lost incompatibil- tained. Genomic DNA from these four transformantsand from the recipient strain was then restricted withity with the het-c3 allele but has retained incompatibility

with the het-c1 and het-c4 antagonistic alleles (Espagne SalI and XhoI and subsequently submitted to Southernblotting, using the 696-bp HindIII fragment as a probeet al. 1997). Our results suggest that amino acids present

at the polymorphic positions in the sixth and the seventh (Figure 5). In the disrupted locus, only the 768-bp SalI-XhoI fragment was expected. The DNA of the recipientrepeats are important for the incompatible interaction

with the het-c3 allele. strain (Figure 5B, lane D) contains both the 768-bpSalI-XhoI and the 1547-bp SalI fragments. These twoDisruption of the het-d2Y allele: het-d2Y gene inactiva-

tion was performed to investigate its potential function fragments were also detected in the DNA of three trans-formants (Figure 5B, lanes 1–3). These transformantsbesides incompatibility. The het-d2Y gene, cloned on a


compatibility, the inactivation of het-d had any othereffect on fungus biology, the phenotype of the �D strainwas investigated under different conditions. The mutantstrain phenotype was found to be similar to the wildtype during its vegetative and sexual phases. Double-mutant strains containing both disrupted het-c and het-dloci were then constructed by crossing single-mutantstrains. The phenotype of these strains was identical tothat of single het-c mutants. They displayed abnormalascospore production but the rate of aborted asci wasnot different from that described for crosses betweensingle het-c mutant strains (Saupe et al. 1994).

The same results were obtained with mutant strainscontaining either a single disrupted het-e locus or the twodisrupted het-c and het-e loci. The lack of any detectablephenotype in strains containing either a disrupted het-e ora disrupted het-d locus may be due to the complementa-tion of one gene by the other one, since they are homol-ogous. To test this hypothesis, double- and triple-mutantstrains containing either disrupted het-e and het-d loci ordisrupted het-e, het-d, and het-c loci were then con-structed. No effect was observed. This result suggeststhat het-e and het-d genes may act only in vegetative in-compatibility.

DISCUSSION

The het-d locus is one of three incompatibility lociFigure 5.—Molecular analysis of strains transformed with involved in the nonallelic incompatibility systems het-c/the pS�D (het-d) disruption vector. (A) Physical map of the

het-e and het-c/het-d. The het-c and het-e loci have been6-kbp SmaI-XbaI fragment containing the het-d2Y gene (at thepreviously characterized (Saupe et al. 1994, 1995). Intop) and of the pS�D disruption construct (at the bottom).

The shaded, solid, and open boxes indicate the DNA encoding this study, we reported the characterization of the vege-the WD40 domain, the intron, and the ura5 gene, respectively. tative incompatibility het-d2Y gene, which encodes a poly-The arrow in the open box gives the direction of gene tran- peptide of 1376 amino acids. As suspected (Espagne etscription. Sizes of the fragments represented by double arrows

al. 1997), sequence comparison indicates that the HET-are given in base pairs. (B) Southern blot analysis of theD2Y polypeptide shows 71% similarity with the HET-E1Agenomic DNA from the recipient strain (lane D) and from four

transformed strains (lanes 1–4). (C) Southern blot analysis of protein (Saupe et al. 1995). Like the HET-E protein,the genomic DNA from the recipient strain (lane D) and from the HET-D putative protein exhibits a putative GTP-the �D knockout mutant strain. Genomic DNAs were digested binding domain in the N-terminal part (Saraste et al.by SalI and XhoI and probed with a 32P-labeled 696-bp HindIII

1990) and a WD40 repeat domain in the C-terminalfragment (B) and with 32P-labeled 1000- and 1547-bp SalI frag-part (Van der Voorn and Ploegh 1992). Moreover,ments (C). The sizes of the fragments are given in base pairs.our results pointed out the presence of a conservedputative incompatibility domain in the N-terminal part.This domain was also described in a N. crassa incompati-probably resulted from complex integration events: in-

sertion by a single crossing over of the pSs�D plasmid bility protein (Smith et al. 2000). Comparison of thishighly conserved domain with sequences present in theprobably duplicated the het-d locus; meanwhile, the resi-

dent het-d gene was inactivated by an unknown mecha- databank reveals a sequence issued from the Neuro-spora sequencing project on chromosome II. It wouldnism. As expected for a perfect gene replacement, the

fourth transformant has lost the 1547-bp SalI fragment be interesting to know if this sequence matches 1 of the11 N. crassa incompatibility loci. Mutational analysesand retained the 768-bp SalI-XhoI fragment (Figure 5B,

lane 4). By probing with the 1000- and 1547-bp SalI on this domain should be performed to confirm theinvolvement of this sequence in the incompatibility ac-fragments, we confirmed that the two SalI fragments,

which were replaced by the ura5 gene in the pS�D tivity. The boxes identified in this domain may definea family of proteins specialized in incompatibilityplasmid, were not detected in the genomic DNA of this

transformant (Figure 5C). This disrupted transformant (Smith et al. 2000). The het-e and het-d gene productsdefine two members of a subfamily of incompatibilitywas called �D and was retained for further studies.

To determine whether, in addition to the loss of in- polypeptides exhibiting, in addition to the incompatibil-


ity specific domain, two other domains: a GTP-binding Concerted evolution of tandemly repeated sequencesis due to gene conversion (Dover 1982) and unequaldomain and a WD40 repeat domain. The HET-E and

HET-D proteins are the only known proteins showing crossing over (Smith 1976). Due to unequal crossingover, the number of repeats is variable among individu-both characteristics of the �- and �-subunits of G-pro-

teins. als as has been shown for the polyubiquitin alleles(Baker and Board 1989). Saupe (2000) proposed thatExcept for vegetative incompatibility, no other func-

tion in the P. anserina life cycle was identified for the the het-e WD40 domain evolved in a concerted way. Thehigh degree of similarity with the HET-D WD40 codinghet-e and het-d genes under laboratory conditions. This

result may be either because the two gene products act sequences suggests that het-d repeats are also subjectedto concerted evolution. The variable number of repeatsexclusively in incompatibility or because another gene

complements the loss of function caused by the inactiva- found in the different het-e and het-d alleles corroboratesthis suggestion. Concerted evolution seems not to betion of the het-e and het-d genes.

We previously reported that the P-loop domain and the only mechanism to explain the het-e and het-d repeatevolution. In fact, five positions are highly polymorphicthe WD40 repeat number of the HET-E protein are

essential for incompatible activity (Saupe et al. 1995; between the WD40 repeats in the HET-E and HET-Dpolypeptides. These polymorphic positions are locatedEspagne et al. 1997). The P-loop sequence is conserved

in the HET-D polypeptide. Depending on the het-e or in the loop between the d and a strands and the turnbetween the b and c strands. Evolution seems to havehet-d active or inactive allele, the number of WD40 re-

peats ranges from 3 to 12 (Espagne et al. 1997) and 6 proceeded in two stages: homogenized concerted evolu-tion followed by specific mutational evolution at specificto 13 in HET-E and HET-D polypeptides, respectively.

Analyses of the different het-d alleles show that all those polymorphic positions.The crystal structure of the G� protein shows that thethat are reactive in incompatibility contain 11 full-length

WD40 repeats and a 12th truncated repeat. The number seven WD40 repeats fold into a seven-blade �-propeller(Sondek et al. 1996). The seven-blade �-propeller hasof WD40 repeats seems to be very important in de-

termining incompatible interaction. Of the 42 amino also been demonstrated for the C-terminal domain ofTup1 (Sprague et al. 2000). The Tup1-Ssn6 complexacids, 33 are conserved within the 10 HET-E1A repeats

(Saupe et al. 1995) and 31 out of the 42 amino acids regulates the expression of several sets of genes in theyeast Saccharomyces cerevisiae. The �-propeller structureare conserved within the 11 HET-D2Y repeats. Whereas

the similarity between the repeats of HET-E1A and HET- looks like a torus with a central tunnel. In the case of ahighly symmetrical structure like the HET-E and HET-DD2Y polypeptides is very strong and the number of WD40

repeats is variable, this is not the case for all other known WD40 repeats, the central tunnel must be quite circular.In WD40 repeat propellers, a hallmark of blades is theWD40 proteins.

Concerted evolution can occur in two different cases: hydrogen-bonded tetrad comprising Trp-30, Thr/Ser-20, His-2, and Asp-24 (Wall et al. 1995; Sondek et al.in multiple copies of a given gene family and/or in

tandemly repeated sequences of a single gene (Smith 1996; Sprague et al. 2000). In the G� subunit and inTup1, not all members of the tetrad are present in every1976). These two different cases of concerted evolution

can apply to the het-e and het-d genes. motif. In the HET-E and HET-D polypeptides, all fourmembers of the hydrogen-bonded structural tetrad areMultiple copies of a given gene family can undergo

concerted evolution so that the sequences of all gene found in each repeat, suggesting that more than sevenblades are involved in the HET-E and HET-D putativecopies are very similar within a given species, although

they normally diverge between different species. The �-propeller structure.The function of the WD40 repeats appears to be theprimary driving force for concerted evolution is intra-

chromosomal; interchromosomal genetic exchanges organized binding of many proteins, either simultane-ously or sequentially (Neer and Smith 2000). The G�are much rarer. The het-e and het-d genes could have

arisen by gene duplication. Stewart and Cullen subunit is tightly bound to the bottom surface of theG� propeller. The G� subunit is bound to the G� central(1999) showed that duplicated genes are often clus-

tered. Recent results indicate that the het-d and het-e tunnel (Neer and Smith 2000). The G�� structure in-teracts with �20 proteins (Clapham and Neer 1997).genes are not found in the same chromosome (C. Bar-

reau, personal communication). Moreover, the amino These interacting proteins bind either to the top surfacenear the central tunnel or to the side of the torus (Neeracid conservation between the HET-E and HET-D WD40

repeats is not always linked to codon conservation: 11 and Smith 2000). Given the conservation of the WD40repeats, it is very likely that all proteins containing multi-conserved amino acids do not use the same codon in

the HET-E and HET-D WD40 repeats (data not shown). ple WD40 repeats will form a propeller structure. Onthe basis that the HET-E and HET-D polypeptides, evenAs the het-d and het-e genes are not in the same chromo-

some and do not seem to be subjected to concerted with more than seven repeats, take a �-propeller struc-ture, the polymorphic positions found in the differentevolution, the het-d and het-e genes seem to be orthologs,

having been subjected to parallel evolution. repeats will all be located at the top of the �-propeller.


Berges, T., and C. Barreau, 1989 Heat shock at an elevated temper-Our results show that amino acids present at the poly-ature improves transformation efficiency of protoplasts from Po-

morphic positions in the sixth and seventh repeats are dospora anserina. J. Gen. Microbiol. 135: 601–604.Bernet, J., 1965 Mode d’action des genes de “barrage” et relationessential for the incompatible interaction between the

entre l’incompatibilite cellulaire et l’incompatibilite sexuelleHET-E2 and HET-C3 proteins. Begueret et al. (1994)chez Podospora anserina. Ann. Sci. Nat. Bot. Ser. 12 6: 611–768.

proposed the poison complex model to explain the Bernet, J., 1967 Les systemes d’incompatibilite chez le Podosporaanserina. C. R. Acad. Sci. Paris 265: 1330–1333.vegetative incompatibility reaction. If the two alleles are

Bourne, H. R., D. A. Sanders and F. McCormick, 1991 The GTPasecompatible, the HET-E (or HET-D) and HET-C proteinssuperfamily: conserved structure and molecular mechanism. Na-

will form a viable complex. If the two alleles are incom- ture 349: 117–127.Bruchez, J. J. P., J. Eberle and V. E. Russo, 1993 Regulatory se-patible, the HET-E (or HET-D) and HET-C proteins

quences in the transcription of Neurospora crassa genes: CAATwill form a poison complex that will be lethal for thebox, TATA box, introns, poly(A) tail formation sequences. Fungal

cell. Although a physical interaction between HET-E Genet. Newsl. 40: 89–96.Clapham, D. E., and E. J. Neer, 1997 G protein beta gamma sub-(or HET-D) and HET-C has not been demonstrated yet,

units. Annu. Rev. Pharmacol. Toxicol. 37: 167–203.it is possible that the HET-C proteins interact at the topCortesi, P., and M. G. Milgroom, 1998 Genetics of vegetative in-

of the HET-E �-propeller. The polymorphism located compatibility in Cryphonectria parasitica. Appl. Environ. Microbiol.64: 2988–2994.at the top of the �-propeller structure should be main-

Coustou, V., C. Deleu, S. Saupe and J. Begueret, 1997 The proteintained to allow binding of HET-C protein from differentproduct of the het-s heterokaryon incompatibility gene of the

het-c alleles. fungus Podospora anserina behaves as a prion analog. Proc. Natl.Acad. Sci. USA 94: 9773–9778.Different hypotheses referring to the biological sig-

Dover, G., 1982 Molecular drive: a cohesive mode of species evolu-nificance of vegetative incompatibilty have been pro-tion. Nature 299: 111–117.

posed (Glass and Kuldau 1992; Begueret et al. 1994; Espagne, E., P. Balhadere, J. Begueret and B. Turcq, 1997 Reac-tivity in vegetative incompatibility of the HET-E protein of theWorrall 1997; Saupe 2000). There are two major con-fungus Podospora anserina is dependent on GTP-binding activityflicting theories. According to the first hypothesis, vege-and a WD40 repeated domain. Mol. Gen. Genet. 256: 620–627.

tative incompatibility limits heterokaryosis to prevent Esser, K., 1974 Podospora anserina, pp. 531–551 in Handbook of Genet-ics, edited by R. C. King. Plenum Press, New York.horizontal transmission of viruses or other deleterious

Esser, K., and R. Blaich, 1973 Heterogenic incompatibility in plantsorganelles. If this is the case, het gene evolution shouldand animals. Adv. Genet. 17: 107–152.

favor maintaining the occurrence of vegetative incom- Fong, H. K., J. B. Hurley, R. S. Hopkins, R. Miake-Lye, M. S. Johnsonet al., 1986 Repetitive segmental structure of the transducin �patibility. In the second hypothesis, however, vegetativesubunit: homology with the CDC4 gene and identification ofincompatibility is considered an accident of evolution.related mRNAs. Proc. Natl. Acad. Sci. USA 83: 2162–2166.

For instance, neutral polymorphism in het genes creates Garcia-Higuera, I., J. Fenoglio, Y. Li, C. Lewis, M. P. Panchenkoet al., 1996 Folding of proteins with WD-repeats: comparisona variant with lethal consequences for the heterokaryo-of six members of the WD-repeat superfamily to the G proteintic cell when it is associated with its incompatible antago-� subunit. Biochemistry 35: 13985–13994.

nist. In the “accident” hypothesis, there is no biological Glass, N. L., and G. A. Kuldau, 1992 Mating type and vegetativeincompatibility in filamentous ascomycetes. Annu. Rev. Phytopa-reason to maintain polymorphism selectively among hetthol. 30: 201–224.genes. Our results, however, demonstrate that the fungi

Glass, N. L., S. J. Vollmer, C. Staben, J. Grotelueschen, R. L.selectively maintain specific polymorphism at the top Metzenberg et al., 1988 DNAs of the two mating-type alleles

of Neurospora crassa are highly dissimilar. Science 241: 570–573.of the �-propeller structure, ruling out the accidentGlass, N. L., J. Grotelueschen and R. L. Metzenberg, 1990 Neu-hypothesis in this case. Vegetative incompatibility seems,

rospora crassa A mating-type region. Proc. Natl. Acad. Sci. USAinstead, to be necessary in the P. anserina life cycle. 87: 4912–4916.

Grindle, M., 1963 Heterocaryon incompatibility of unrelatedE.E. was supported by a fellowship from the Ministere de la Re-strains in the Aspergillus nidulans group. Heredity 18: 191–204.cherche.

Javerzat, J. P., C. Jacquier and C. Barreau, 1993 Assignment oflinkage groups to the electrophoretically-separated chromosomesof the fungus Podospora anserina. Curr. Genet. 24: 219–222.

Lambright, D. G., J. Sondek, A. Bohm, N. P. Skiba, H. E. Hamm et al.,LITERATURE CITED1996 The 2.0 A crystal structure of a heterotrimeric G protein.Nature 379: 311–319.Akins, R. A., and A. M. Lambowitz, 1985 General method for clon-

ing Neurospora crassa nuclear genes by complementation of mu- Lecellier, G., and P. Silar, 1994 Rapid methods for nucleic acidsextraction from Petri dish-grown mycelia. Curr. Genet. 25: 122–tants. Mol. Cell. Biol. 5: 2272–2278.

Anagnostakis, S. L., 1977 Vegetative incompatibility in Endothia 123.Loubradou, G., and B. Turcq, 2000 Vegetative incompatibility inparasitica. Exp. Mycol. 1: 306–316.

Baker, R. T., and P. G. Board, 1989 Unequal crossover generates filamentous fungi: a roundabout way of understanding the phe-nomenon. Res. Microbiol. 151: 239–245.variation in ubiquitin coding unit number at the human UbC

polyubiquitin locus. Am. J. Hum. Genet. 44: 534–542. Neer, E. J., and T. F. Smith, 2000 A groovy new structure. Proc.Natl. Acad. Sci. USA 97: 960–962.Ballance, D. J., 1991 Transformation systems for filamentous fungi

and an overview of fungal gene structure, pp. 1–29 in Molecular Orbach, M. J., 1994 A cosmid with a HyR marker for fungal libraryconstruction and screening. Gene 150: 159–162.Industrial Mycology, edited by S. A. Leong and R. M. Berka. Marcel

Dekker, New York. Perkins, D. D., A. Radford, D. Newmeyer and M. Bjorkman, 1982Chromosomal loci of Neurospora crassa. Microbiol. Rev. 46: 426–Begueret, J., B. Turcq and C. Clave, 1994 Vegetative incompatibil-

ity in filamentous fungi: het genes begin to talk. Trends Genet. 570.Rizet, G., 1952 Les phenomenes de barrages chez Podospora anse-10: 441–446.

Beisson-Schecroun, J., 1962 Incompatibilite cellulaire et interac- rina: analyse genetique des barrages entre les souches s et S. Rev.Cytol. Biol. Veg. 13: 51–92.tions nucleo-cytoplasmiques dans les phenomenes de “barrage”

chez le Podospora anserina. Ann. Genet. 4: 3–50. Rizet, G., and C. Engelman, 1949 Contribution a l’etude genetique


d’un ascomycete tetraspore: Podospora anserina. Rev. Cytol. Biol. Smith, T. F., C. Gaitatzes, K. Saxena and E. J. Neer, 1999 TheVeg. 11: 201–304. WD repeat: a common architecture for diverse functions. Trends

Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular Clon- Biochem. Sci. 24: 181–185.ing: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Sondek, J., A. Bohm, D. G. Lambright, H. E. Hamm and P. B. Sigler,Press, Cold Spring Harbor, NY. 1996 Crystal structure of a G-protein �� dimer at 2.1A resolu-

Saraste, M., P. R. Sibbald and A. Wittinghofer, 1990 The P-loop, tion. Nature 379: 369–374.a common motif in ATP- and GTP-binding proteins. Trends Sprague, E. R., M. J. Redd, A. D. Johnson and C. Wolberger, 2000Biochem. Sci. 15: 430–434. Structure of the C-terminal domain of Tup1, a corepressor of

Saupe, S. J., 2000 Molecular genetics of heterokaryon incompatibil- transcription in yeast. EMBO J. 19: 3016–3027.ity in filamentous ascomycetes. Microbiol. Mol. Biol. Rev. 64: Staben, C., and C. Yanofsky, 1990 Neurospora crassa a mating-type489–502. region. Proc. Natl. Acad. Sci. USA 87: 4917–4921.

Saupe, S., C. Descamps, B. Turcq and J. Begueret, 1994 Inactiva- Stewart, P., and D. Cullen, 1999 Organization and differentialtion of the Podospora anserina vegetative incompatibility locus het-c, regulation of a cluster of lignin peroxidase genes of Phanerochaetewhose product resembles a glycolipid transfer protein, drastically chrysosporium. J. Bacteriol. 181: 3427–3432.impairs ascospore production. Proc. Natl. Acad. Sci. USA 91: Turcq, B., and J. Begueret, 1987 The ura5 gene of the filamentous5927–5931.

fungus Podospora anserina : nucleotide sequence and expressionSaupe, S., B. Turcq and J. Begueret, 1995 A gene responsiblein transformed strains. Gene 53: 201–209.for vegetative incompatibility in the fungus Podospora anserina

Turcq, B., M. Denayrolles and J. Begueret, 1990 Isolation of theencodes a protein with a GTP-binding motif and G� homologoustwo allelic incompatibility genes s and S of the fungus Podosporadomain. Gene 162: 135–139.anserina. Curr. Genet. 17: 297–303.Saupe, S. J., G. A. Kuldau, M. L. Smith and N. L. Glass, 1996 The

van der Voorn, L., and H. L. Ploegh, 1992 The WD-40 repeat.product of the het-C heterokaryon incompatibility gene of Neuro-FEBS Lett. 307: 131–134.spora crassa has characteristics of a glycine-rich cell wall protein.

Wall, M. A., D. E. Coleman, E. Lee, J. A. Iniguez-Lluhi, B. A. PosnerGenetics 143: 1589–1600.et al., 1995 The structure of the G protein heterotrimer Gi�1�1�2.Smith, G. P., 1976 Evolution of repeated DNA sequences by unequalCell 83: 1047–1058.crossover. Science 191: 528–535.

Smith, M. L., O. C. Micali, S. P. Hubbard, N. Mir-Rashed, D. J. Worrall, J. J., 1997 Somatic incompatibility in basidiomycetes. My-Jacobson et al., 2000 Vegetative incompatibility in the het-6 cologia 89: 24–36.region of Neurospora crassa is mediated by two linked genes. Genet-ics 155: 1095–1104. Communicating editor: R. H. Davis

Documents

HET-E and HET-D Belong to a New Subfamily of WD40 Proteins Involved in Vegetative Incompatibility Specificity in the Fungus Podospora anserina