Copyright 0 1985 by the Genetics Society of America

A NEW METHOD FOR T H E SCREENING O F MUTATIONS RELATED TO PROTOPLASMIC INCOMPATIBILITY,

DIFFERENTIATION AND PLASMA MEMBRANE IN T H E FUNGUS PODOSPORA ANSERINA

JACQUES LABARERE A N D MARC BONNEU

Laboratoire de Ginnktique Moliculaire, Universiti de Bordeaux II, I.N.R.A., Domaine de la Grande Ferrade, 33140 Pont de la Maye, France

Manuscript received January 3 1, 1985 Revised copy accepted May 16, 1985

ABSTRACT

In Podospora anserina previous investigations showed that mutations in genes involved in the control of protoplasmic incompatibility cause defects at various stages of differentiation during the life cycle and also modify properties of the plasma membrane. To establish these relationships in another way, a new method for screening mutations has been developed as a first step. Eighty-five new mutants were selected for resistance to toxic products (sorbose or thi- ourea); in a second step these mutants were tested for modifications of proto- plasmic incompatibility and cellular differentiation. Seven of the sorbose or thiourea resistant-mutants (i.e., 8%) exhibited new patterns of protoplasmic incompatibility. Genetic analyses were carried out with three mutants. Mutation X25 suppresses protoplasmic incompatibility resulting from all allelic interac- tions and restores the fertility of the crosses 0 V X B V I and 0 ZI x 8 22. Mutation V41 is an allele of the v locus with new properties. Mutation X61 totally suppresses the V/V’l interaction and weakens all of the other allelic incompatibility systems; X61 strains are defective in protoperithecia differentia- tion. Electrophoresis of plasma membrane proteins showed that X61 strains lack two polypeptides whose apparent molecular weights are 41,000 and 44,000. This new screening method is especially efficient for obtaining new mutants and identifying additional genes involved in incompatibility. These results provide further support demonstrating the relationships between pro- toplasmic incompatibility, cellular differentiation and plasma membrane.

ROTOPLASMIC, vegetative or heterogenic incompatibility is a common feature in fungi, higher plants and animals. In fungi, it has been reported

among Neurospora crassa (PERKINS and BARRY 1977), Podospora anserina (Es- SER 1954; LABARERE and BERNET 1979a), Endothia parastica (ANAGNOSTAKIS 1983), the genus Aspergillus @ALES, MOORHOUSE and CROFT 1983), Physarum polycephalum (SCHRAUWEN 1979), Fusarium oxysporum (SANCHEZ, LEARY and ENDO 1976) and the myxomycete Didymium iridis (LING and CLARK 1981).

Many hypotheses have been proposed to explain the role and mechanism of protoplasmic incompatibility in fungi. CATEN (1 972) proposed that proto- plasmic incompatibility might serve as a defense mechanism protecting the

Genetics 111: 259-271 October, 1985.

260 J. L A B A R ~ R E AND M. BONNEU

TABLE 1

Resistance and sensitivity of N-methyl-N ‘-nitro-N-nitrosoguanidine-induced mutant strains to sorbose, thiourea and methylammonium

Growth on

Basic Basic Basic medium +

Class Mutant strains medium sorbose thiourea ammonium Basic medium + medium + methyl-

A

B

C

D

E F G H I

K L M

J

72, 73, 74, 75, 76, 77 , 78, 81, 82, 83, 84

1, 4, 10, 14, 15, 29, 40, 48, 49, 50, 53, 56, 57, 62, 63, 65, 68

5, 16, 18, 21, 35, 46, 52, 54, 66, 69, 70

9, 23, 34, 36, 37, 42, 44, 45, 51, 58, 60

71, 79, 80 13, 19 1 1 , 20, 39, 61, 64 25, 26 12, 24, 41 2, 7, 28, 32, 47 8, 27, 31, 55 22, 30, 67 3, 6, 17, 33, 38, 43, 59

<

R

HR

R

S R R HR HR HR R R HR

HR

HR R S R S - - - - S S

S, =, R and HR designate growth rate compared to wild type on supplemented media, respec- tively: 60-90, 100, 110-200 and more than 200%; < designates a mutant growth lower than wild type on basic medium.

organism against cytoplasmic invasion. ESSER and BLAICH (1 973) proposed that heterogenic or protoplasmic incompatibility has a role of restricting outbreed- ing. HARTL, DEMPSTER and BROWN (1975) proposed that in N. crassa incom- patibility prevents the spread of nonadaptative heterokaryon exploitive nuclei. LABARERE and BERNET (1979a) proposed that in P. anserina protoplasmic in- compatibility is due to the pathological effect of specific lytic proteases whose deregulated expression is related to modifications of plasma membrane pro- teins (ASSELINEAU, BERNET and LABARERE 198 1 ; BONNEU and LABARERE

In the ascomycete P. anserina protoplasmic incompatibility occurs during heterokaryosis and leads to cell destruction resulting from the association of the two incompatible genes in cells joined. Protoplasmic incompatibility has been shown to be under the control of many genes that determine allelic or nonallelic systems (BERNET 1965). Allelic incompatibility systems involve genes of four loci (U, 6 , q , z ) and nonallelic systems genes of five loci ( r , U, c, d , e) . The U locus functions both allelically and nonallelically (BERNET 1965; LABAR- ERE 1973). Protoplasmic incompatibility is completely alleviated by mutations in mod genes (LABARERE and BERNET 1978, 1979b). Previous results have shown that the mod genes control the production of specific proteases (BE-

1983).

MUTATIONS IN P O m S P O R A ANSERINA

TABLE 2

Effect of mutations on protoplasmic incompatibility: results of confrontations between the mutant strains and tester strains of known genotypes

26 1

~~~~~ ~

Tester strain genotype"

V I R

Original strain

Mutant strain Class I

25 61

Class I1 41 67

Class 111 63 83 a4

A I 1 I I I I

A W W W W W I A W W W A W I

A I I I A I I A 1 I 1 W W I

A W W W I I I A W W W I I I A I I W I 1 1

I I

I I

I I

I I I I

I I I I I I

The interaction involved in each confrontation is indicated in brackets. The presence, weak- ening or absence of a barrage in confrontation is, respectively, indicated by the letters I (incom- patibility, W (weakened incompatibility) or A (absence of incompatibility).

With respect to the protoplasmic incompatibility genes, the genotype of a strain involves the b, q, z, U, r, c, d , e loci and the genotype of the mutagenized strain was c(S) d(S) e@) V r B l Ql 21. To simplify nomenclature of the tester strains, only the genes that are different from the wild- type original strain are indicated.

GUERET and BERNET 1973; LABARERE 1980) and the differentiation stages of the life cycle (LABARERE and BERNET 1979a,b). Biochemical studies have sug- gested that some of these incompatibility-related genes might code for intrinsic proteins of the plasma membrane (ASSELINEAU, BERNET and LABARERE 198 1). This hypothesis was recently demonstrated by studies of plasma membrane proteins from the mod c mutants (BONNEU and LABARERE 1983).

Major difficulties in studying protoplasmic incompatibility and developing experimental support for the above hypotheses reside in screening for muta- tions and obtaining new incompatibility alleles and genes. Therefore, we ex- tended our investigations toward the use of mutants that were selected for their strongly modified sensitivity to toxic products. The screening method is based on the already known properties of the mod c mutants, namely, the alteration of their pattern of sensitivity to toxic products.

In P. anserina all previous methods were based upon the selection of strains in which incompatibility was suppressed; these techniques gave only a small variety of mutants. The screening procedure described in this paper is original in that it selects mutants on the basis of their altered sensitivity to toxic prod- ucts, after which we study in these mutants the modifications of the incom- patibility pattern and of the plasma membrane protein pattern. This has al-

262 J. LABAR~RE AND M. BONNEU

lowed us to demonstrate the relationships among plasma membrane, differ- entiation and incompatibility.

These mutants were then used to study the modification of the incompati- bility pattern in relation to the changes in patterns of the membrane proteins.

MATERIALS AND METHODS

Organism: P. anserina is a heterothallic ascomycete whose life cycle closely resembles that of N . crassa. The only well-differentiated structure is the female organ (protoperithecium). Asci contain four binucleated spores that include both products of a half tetrad. In about 5% of asci, one binucleated spore is replaced by two uninucleated spores. The homokaryotic strains are obtained by germination of the uninucleated spores which produce the homokaryotic strains (+ or -), and it is these isolates that are used in our experiments for genetic analyses. For details of the life cycle see BSER (1974).

Strains and incompatibility gene nomenclature: The reference wild-type strain used was the geo- graphical race designated (Cs). Alleles of the c , d , e, r , U, b, q and L loci give rise to three nonallelic incompatibility systems (C/D, C/E, R/V) and to four allelic incompatibility systems (V/VI, BI/B2, Ql/Q2, 21/22) . At the c, d and e loci, studies of 16 geographical races called (A), (B), (C), etc. revealed allelic series (BERNET 1965); c(S), d(A) , etc. designate c and d alleles from the geographical races (S) and (A). At the r, b, q and z loci only two alleles were known: r and R , B l and 82, Ql and Q2, Z1 and 22. At the v locus two wild-type alleles (V and V I ) and one mutant allele ( V ' I ) have been identified (LABARERE 1973). The v gene intervenes both in allelic (V/VI and V/V'l) and nonallelic (V/R and V'l/R) interactions. So a V strain is incompatible with the V I , V ' l and R strains; and a V'I strain is incompatible with the V1 and R strains. The VR and V ' l R strains, which combine the R and V incompatible genes, are lethal.

Medium and culture conditions: Natural medium containing cornmeal extract and malt extract (ESSER 1974) was used for growth and was supplemented with sorbose (3 mg/ml) or thiourea (3 mg/ml) or methylammonium (1.5 mg/ml) to constitute media for mutant selection. Ammonium acetate (4 mg/ml) was added to the basic natural medium for ascospore germination and dihydro- streptomycin (6 mg/ml) to perform crosses. The standard temperature for spore germination, mycelial growth, protoperithecium formation and perithecial development was 26".

Barrage confrontations for protoplasmic incompatibility: T h e term "barrage" designates an unpig- mented area formed on the confrontation line between two strains of incompatible genotypes. In the barrage area, mixed cells formed by the fusion of hyphae of the two confronted strains are destroyed by a lytic process (RIZET and SCHECROUN 1959; BERNET 1965). To assay for protoplasmic incompatibility, strains were cultivated on basic natural medium in Petri dishes for 4 days at 26" in the dark. T h e presence of a barrage on the confrontation line indicates a protoplasmic (or vegetative or heterogenic) incompatibility reaction.

N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis: For mutagenesis the reference wild-type strain, whose genotype was c(S) e(S) d(S) V r B1 QI 21, was grown for 2 days on cellophane pads deposited on basic natural medium. Then, the cellophane pads were transferred into a solution containing 50 pg/ml of N-methyl-N'-nitro-N-nitrosoguanidine and 9 mg/ml of NaCl for 10 min, washed three times with a solution containing 9 mg/ml of NaCl and returned to the natural medium supple- mented with sorbose or thiourea. On these media growth of wild type was completely stopped and sorbose- or thiourea-resistant strains were isolated from sectors in which growth resumed,

Isolation of plasma membranes: Plasma membranes were prepared as previously described (LA-

Two-dimensional polyacrylamide gel electrophoresis of plasma-membrane: Proteins of plasma mem- brane preparation were solubilized in a sodium dodecyl sulfate (SDS) sample buffer [2% (w/v) SDS, 10% (w/v) glycerol, 5% (v/v) P-mercaptoethanol, 0.0625 M Tris-HCI, pH 6.81. Subsequently, two volumes of a solution consisting of 9.5 M urea, 2% (w/v) ampholines (0.4%, pH 3.5-10; 1.6%, pH 5-7), 5% (v/v) P-mercaptoethanol and 8% (w/v) NP-40 were added. Sample volume was usually less than 80 PI and contained at least 30 r g of protein.

In the first dimension, isoelectric focusing procedure was performed according to O'FARRELL

BARERE and BONNEU 1982).

MUTATIONS IN POLWSPORA ANSERINA 263 (1975). After electrophoresis, first dimension gels were equilibrated for 30 min in the SDS sample buffer prior to electrophoresis in the second dimension.

In the second dimension, SDS-gel electrophoresis was performed as described by O'FARRELL (1975) using a 10% acrylamide-separating gel with a 4% stacking gel. The second dimension SDS- polyacrylamide gels were stained using the rapid, sensitive silver strain of MERRIL, DUNAU and COLDMAN (1 98 1).

RESULTS

Sensitivity and resistance of the mutants to toxic products: Following mutagenesis of the wild-type strain, 70 sorbose-resistant and 15 thiourea-resistant mutants were obtained. To test their sensitivity patterns to toxic products, each mutant strain was picked out into Petri dishes containing natural media supplemented with sorbose, thiourea or methylammonium. Growth rates, i.e., millimeter in- crease in diameter of strains, were determined by measuring diameters after 36, 60 and 80 hr of growth at 26". Growth rate on the basic natural media was also measured. On each Petri dish, the wild-type strain constituted the reference. According to this criterion four categories of growth rates were defined: sensitive, identical, resistant and hyperresistant, whose growth rates were, respectively, 60-90, 100, 110-200 and more than 200% of wild type. Results are summarized in Table 1. Mutants were distributed into 13 classes, two (A and E) were thiourea hyperresistant and 11 were sorbose resistant. Four classes (A, B, C and D) were modified for sensitivity to one toxic product and all of the other classes to two toxic products.

Effect of the mutations on protoplasmic incompatibility: It was hypothesized that modifications of the drug sensitivity may result, in some cases, in mutations in incompatibility genes or in genes whose functions were closely related to the protoplasmic incompatibility reaction. Each mutant strain was tested for mod- ifications of protoplasmic incompatibility in confrontations with tester strains of known genotypes. The genotype of the progenitor of the mutants was c(S) d(S) e(S) V r B l Ql 21. Therefore, the nonallelic interactions c(S)/e(A), c(S)/ d(A) and V/R and the allelic ones V/V1, V/V'1, Bl/B2, Ql/Q2 and 21/22 were investigated (Table 2). Modifications of protoplasmic incompatibility re- sulted in the disappearance or weakening of the barrage between mutant and tester. Seven mutants ( 2 5 , 41, 61, 63, 67, 83, 84) were modified in their reactions. We have distributed these mutants in three classes (I, 11, 111). In class I (mutants 25 and 61) all of the allelic interactions were weakened or suppressed. In class I1 (mutants 41 and 67) only interactions determined by the v gene (V'l /V and Vl/V) were modified and in class I11 (mutants 63, 83 and 84) only interactions resulting from the b, q or z genes were modified.

Effect of the mutations on sexual incompatibility: Like protoplasmic incompati- bility, sexual incompatibility results from the interaction of two genes. It occurs during the merging of the male gamete with the trichogyne of the female organ. This prevents fertilization and the subsequent development of the fruit- ing bodies (perithecia). Before mutagenesis, compared to the fertile reference crosses 9 V r X d V r or 0 V l r X d V1 r , the original strain showed strong sexual incompatibility with the VI R, c(A) e(A) and c(A) d(A) strains used as female parents (Le., based on the d V x 0 R , d c(S) X 0 e@) and d c(S) X ?

264 J. LABARERE AND M. BONNEU

TABLE 3

Sexual incompatibility determined by perithecial density after fertilization of mutant strains by tester strains carrying various incompatibility genes

Tester strains ~

6 V I P R 9 4'4) 0 44 Strain [VIV 11 [VIR1 [c(S)Ie(A)l [c(S)/d(A)I

Original strain 2 x lo-' 2 x 10-4 5 x 10-4 14 X lo-' Mutant strains

41 1 2 x 10-4 5 x 1 0 - ~ 14 X lo-' 25 1 2 x 10-4 5 x 10-4 14 X lo-' Other 2 x 10- 2 x 1 0 - ~ 5 x 1 0 - ~ 14 X lo-'

Incompatible interactions are indicated in brackets. Numbers indicate the relative fertility, rep- resented by perithecial density, compared to a 100% fertile reference cross. Perithecial density was determined 5 days after fertilization, counting the number of perithecia observed with a binocular magnifying glass on five 1-cm2 areas in Petri dishes.

TABLE 4

Protoperithecial density of mutant and wild-type strains

Protoperithecial density Strains (an-')"

Wild-type 1000 Mutant-strains

10, 15, 61, 66, 70 0 50-1 50 1 , 23, 50, 56, 58, 84

Strains were grown on natural medium for 5 days in the dark and exposed to light for 8 days after the mycelia have reached the edge of Petri dish.

Protoperithecial density was then determined counting the number of protoperithecia observed with a binocular magnifying glass on five 1-cm2 areas in each Petri dish.

d(A) interactions) and weak sexual incompatibility with the V1 r strain used as male parent (i.e., based on the 0 V X d V I interaction). In regard to sexual incompatibility two mutant strains exhibited modified fertility (Table 3). When strains 25 and 41 were used as female parents in crosses with a V l r strain these mutant strains displayed normal fertility; thus, the partial sterility result- ing from the interaction of the V and V1 alleles was totally suppressed.

Effect of the mutations on protoperithecia formation: Mutations modifying pro- toplasmic incompatibility can also affect the development of protoperithecia (LABARERE and BERNET 1977, 1978, 1979a,b). The ability of the mutant strains to produce protoperithecia was compared to mycelia after 8 days of light exposure at 26". Eleven mutant strains showed reduced density of pro- toperithecia formation (Table 4). Five (10, 15, 61, 66, 70) were unable to produce protoperithecia and six (1, 23, 50, 56, 58, 84) had a low density of protoperithecia, ranging from 5 to 15% of the wild-type density. Mutants 61 and 84 were modified for protoplasmic incompatibility as well as protoperithe- cia differentiation.

MUTATIONS IN PODOSPORA ANSERINA 265

Mutant strains used for genetic analyses: It was previously shown that some mutations screened for suppression of incompatibility induced also modifica- tions in the sensitivity to toxic products (ASSELINEAU, BERNET and LABARERE 198 1) in protoperithecia development (LABARERE and BERNET 1977) and in composition of plasma membrane proteins (BONNEU and LABARERE 1983). Therefore, we extended our investigations and genetic analyses using the mu- tants that were screened for modification of the toxic sensitivity, and then, we characterized their incompatibility pattern and differentiation modifications. Mutants 25, 41 and 61 are modified for the majority of these characters. All were screened for modified sensitivity to sorbose and thiourea and were also modified for protoplasmic incompatibility. In addition, mutants 41 and 25 were modified for sexual incompatibility and mutant 6 1 was unable to differentiate female sexual organs.

Results of crosses between the mutant strains and the original wild-type strain: To determine whether the pleiotropic phenotype resulted from one or more mutations, these mutants were crossed with the original wild-type strain. Anal- ysis of segregation (data not included) shows that the pleiotropic phenotype of mutant 61 resulted from a single lesion. The mutated gene was called X 6 1 . This single mutation was responsible for the sorbose resistance, the thiourea sensitivity, the absence of protoperithecia, the suppression of the V/V' 1 and the weakening of the Vl/V, Bl/B2, Ql/Q2, 21/22 interactions.

Segregation analysis (data not included) indicates that the mutant 25 phe- notype resulted also from a mutation in a single gene named X 2 5 . This mu- tation was responsible for the sorbose hyperresistance, the thiourea resistance and the weakening of the protoplasmic incompatibility resulting from all allelic interactions.

Progeny from the cross between the mutant 41 and the original wild-type strain were equally distributed in two classes of protoplasmic incompatibility (data not included), indicating that the determinant of altered protoplasmic incompatibility was due to a single mutation designated X 4 1 . A surprising element is that, with respect to the toxic product sensitivity, all of the progeny possessed wild-type phenotypes. The results were identical when the mutant was used as male or as female. Nevertheless, the sorbose hyperresistance and the thiourea sensitivity persisted in the original culture and were maintained after 1 yr of vegetative growth at 26" and after 1 yr of storage at 4". At this time, we have no explanation for the noninheritance of the sorbose hyperres- istance and for the thiourea sensitivity by means of sexual reproduction.

Fertility was studied in the progeny of the crosses of mutants 41 and 25 with the wild-type strain. From each progeny, 20 mycelia of wild-type and mutant phenotypes were recovered and crossed as females with male strains of V 1 genotype; the development of the fruiting bodies was compared to that of reference crosses (VI X V I or V x V ) . All of the wild-type progeny had a relative fertility of 2 X lo-*; all of the mutant progeny had a relative fertility of 1.0. This demonstrates that the X 2 5 and X 4 1 mutations are suppressors of the partial sterility.

Genetic localization and properties of the mutation X4 1 : When mutant 41,

266 J. L A B A R ~ R E AND M. BONNEU

TABLE 5

Progeny of crosses between mutant 41 used as male and a V1 R strain

Presence of a barrage in the confrontation with strains of genotypes

Class No. of strains % v r V' I r V I r Genotypes

A 47 23.5 A A I V41 r B 55 27.5 I A A VI r C 49 24.5 Self-lysing strains V41 R D 49 24.5 I I A VI R

The presence o r the absence of a barrage in strain confrontations is indicated by the letters I (incompatibility) and A (absence of barrage).

TABLE 6

Progeny of crosses between a V1 R X25+ and V1 r X25 strains

Barrage in the confrontations

with V ' l r strains (incompat-

No. o f ibles with R Sorbose Class strains % strains) resistance Genotypes

VI R X25' - - A 101 49.75 I B 93 45.8 A R VI r X 2 5 C 3 1.47 1 R VI R X 2 5

VI r X25' D 6 2.95 A - - I , incompatible; A, absence; R, resistant strain; =, no resistant strain.

whose original genotype was V r, was crossed with a strain of genotype VI R , the progeny were equally distributed in four classes with respect to incompat- ibility reactions (Table 5): classes A and D were parental; B and C were recombinant. Classes A and C possess a new allelic form of the v gene, which we called V41 . This mutant allele derived from the wild-type V allele expressed a new phenotype; it lost its incompatibility reaction with the V'I allele and its sterility when crossed with a male strain carrying the VI allele (Table 3).

Genetic localization and properties of the mutation X25: Mutation X 2 5 weak- ened protoplasmic incompatibility as a result of the interaction in all allelic incompatibility systems (Table 2).

To investigate the genetic localization of the mutation with regard to the r gene, VI r X25 strains obtained from the cross 8 VrX25 X 0 VIrX25+ were crossed with VI R X25+ strains. Progeny confronted with a V'I r strain incom- patible with the VI R strains (V'l /R nonallelic interaction) and tested for sorbose resistance could be separated into four classes corresponding to the parental V I R X25+ and VI r X25 and to the recombinant VI R X25 and VI r X25' genotypes (Table 6) . The results clearly indicate that the X 2 5 mutation is closely linked to the r locus (about four recombination units).

Confrontations between VX25 and V'I X25 , V X25 and VI X 2 5 , BI X25 and B2 X25 , 41 X25 and Q2 X25 and Z I X25 and 22 X25 reveal that the barrage resulting from the allelic interactions V/V' l , V/V1, B1/B2, Ql /Q2 or 21/22

MUTATIONS IN PODOSPORA ANSERINA 267

was totally suppressed if the X25 mutation was present in both strains. Similar confrontations between VI RX25 and V’l r X25, c(S) c(S) X25 and c(A) e(A) X25 and c(S) d(S ) X25 and c(A) d (A) X25 strains showed that the nonallelic interactions V’l/R, C/E and C/D were not suppressed by the X25 mutation.

Genetic localization and properties of the mutation X6 1 : Genetic analysis showed that the X61 mutation was genetically independent of the mating-type locus and of the b, q , z, r , U, c, e and d incompatibility loci. Mutation X61 suppressed the allelic interaction V/V’l when it was present in only one of the two strains confronted. Confrontations between strains V X61 and VI X61, B l X61 and B2 X61, Ql X61 and Q2 X61, Z l X61 and 22 X61 showed no differences from the confrontations V X61 and V I , V and V l X61, etc. The mutation X61 weakened the barrage resulting from these allelic interactions when it was present in one or in both strains confronted.

Two-dimensional electrophoresis of proteins solubilized f rom pur$ed plasma mem- branes f rom wild-type, X25, X41 and X61 mutant strains: Two-dimensional is- oelectric focusing SDS-polyacrylamide gels of proteins solubilized from purified wild-type and X61 membranes are shown in Figure 1 . The X61 mutant strain revealed a constant difference in the plasmalemma protein pattern compared with the original wild-type strain. The X61 strain lacked two spots of apparent molecular weights of 44,000 and 41,000 and of PI = 6.9 (spots E41 and E44). The mutant strains X41 and X25 showed plasmalemma protein patterns iden- tical with the wild-type pattern.

DISCUSSION

It was previously hypothesized that protoplasmic incompatibility involves plasma membrane proteins (ASSELINEAU, BERNET and LABARERE 198 1). This hypothesis was first demonstrated for the modc mutations (BONNEU and LA- BARERE 1983) which suppress the protoplasmic incompatibility caused by the interaction of two specific alleles of the two different loci r and U. Mod c mutations were also modified for protoperithecial differentiation and for sor- bose sensitivity. To demonstrate by another method the relationship among protoplasmic incompatibility, plasma membrane proteins and differentiation we have screened mutants modified for resistance to toxic products and then investigated their modifications related to these phenomena.

Seventy mutants for sorbose resistance and 15 for thiourea resistance were screened for their resistance spectra and investigated for modifications of pro- toplasmic and sexual incompatibility, protoperithecia formation and, for some of them, their plasma membrane proteins. For 40% of the mutants, sensitivity for more than one toxic product was modified; for example, sorbose-resistant mutants were also thiourea resistant or thiourea sensitive. It was demonstrated that the pleiotropic phenotype resulted from a single mutation in mutants 2, 13, 22, 25, 27, 33, 61, 67 and 71 (all data not included). Thiourea and sorbose have different targets in the cell; therefore, when a single mutant was modified for sensitivity to these products, it was possible that the gene affected by mutation might be related to plasma membrane permeability.

Seven mutants having different phenotypes expressed new modifications in

268 J. LABARERE AND M. BONNEU

A B M .W.

- * O K - - 70K - st - 60K -

- 50K -

- 40K -

I I I I 1 I I I I I

G F E D C G F E D C

FIGURE 1 .-Two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis of plasma membrane proteins from wild type (A) and X61 mutant (B). Each sample (80 pl) contained about 30 pg of plasma membrane proteins. For the first dimension, gels were prerun at 200 V for 15 min, 300 V for 30 min. 400 V for 30 min and run at 400 V for 14 hr (constant voltage) and finally at 800 V for 1 hr at room temperature. For the second dimension, a 10% acrylamide separating gel and a 4% stacking gel were used and run at 4 W for 4 hr (constant power). Silver nitrate staining was used. Capital letters correspond to pH zones as defined by BONNEU and LARARERE (1983). Arrows and circle mark the proteins absent in the X61 mutant. M. W., molecular weight in kilodaltons. IF, isoelectric focusing.

the protoplasmic incompatibility pattern (Table 7). One had deficient proto- perithecium differentiation (mutant 6 1) and two had suppression of sexual incompatibility resulting from the allelic interaction V/V 1 (mutants 25 and 41). The screening method described in this paper gives an exceptionally high percentage (8%) of mutants modified for allelic incompatibility; all of the phe- notypes obtained are new. It should be noted that in P. anserina mutations modifying the allelic systems are very rare. Previously, only one mutation of the v locus (about 10,000 isolates screened in the R/V system) had been obtained (LABARERE 1973).

Three mutants (25, 41 and 61) were used for genetic analysis. The X25 mutation restored a total fertility in the sterile cross 9 V X 8 VZ and weakened the barrage resulting from the allelic incompatible interactions V/Vl , V/V' 1, B1/B2, 4 1 / 4 2 and 21/22. This mutation totally suppressed the barrage when it was present in the two strains confronted. The X25 gene has been located at a distance of four recombination units from the r incompatibility locus.

The V4Z mutation is a new allelic form of the v gene. I t is derived from

MUTATIONS IN PODOSPORA ANSERINA 269 TABLE 7

Characteristics of the wild-type and mutant strains modij?ed for protoplasmic incompatibility

Protoplasmic incompatibility interactions Sensitivity to toxic products Protoperi- Fertility

thecial of the cross Strains Sorbose Thiourea density (cm-’) P V X d V1 B1/B2 4 1 / 4 2 21/22 V/Vl V/Vl’

Original strain Mutant 25 Mutant 41 Mutant 61 Mutant 63 Mutant 67 Mutant 83 Mutant 84

- - R S S

S HR HR

- -

1000 1000 1000

0 1000 1000 1000 1000

2 x I I I 1 1 1 w w w w w 1 1 I I I A Not tested W W W W A 2x10-2 w w w I I 2 x lo-* 1 I I W W 2x10-2 w w w I 1 2 x 10-2 I I W I I

S, =, R and HR designate growth rate compared to wild type on supplemented media, respec- tively: 60-90, 100, 100-200 and more than 200%. W, weakened incompatibility; I, incompatibility.

the wild-type V allele, which is incompatible with strains carrying the VI and V ’ l alleles and with strains carrying the nonallelic R gene. Strains carrying the new V4Z allele lost their incompatibility with the V ‘ l allele and had a normal fertility when crossed as female with VI strains (a wild-type V strain is sterile in a similar cross).

The X61 mutation was unable to differentiate protoperithecia. It suppressed the barrage resulting from the allelic interaction V/V’l when it was present in V or V ’ l strains and weakened all of the other allelic interactions when it was present in one or both strains confronted. The X61 gene segregated in- dependently from the other incompatibility genes.

When we compared the plasma membrane proteins of the mutant and wild- type strains, it appeared that the X6Z mutation modified the original protein pattern. X61 strains had lost plasma membrane polypeptides E4 1 (apparent molecular weight = 41,000 and PI = 6.9) and E44 (apparent molecular weight = 44,000 and PI = 6.9). The X61 mutant is genetically distinct from the modc locust (data not included), but its phenotype is similar to that of the modc mutant. We have previously demonstrated that the modc mutations suppressed the protoplasmic incompatibility resulting from the nonallelic V/R interaction and did not differentiate protoperithecia. X61 strains are also unable to differ- entiate protoperithecia and suppress the allelic V/V’ 1 interaction in which the Y loci is involved.

In our earlier investigations on protoplasmic incompatibility we developed the argument that the products of genes involved in incompatibility would also be to plasma membrane proteins and involved in differentiation. This inter- pretation is confirmed for one ( i e . , X61) of the mutants examined in this study.

A number of different systems have been used to select mutants of incom- patibility genes in P. anserina. They were always based on outgrowth from forced heterokaryons containing conditional lethal genotypes or from homo- karyons in which two allelic incompatibility genes were joined in the same

270 J. L A B A R ~ R E AND M. BONNEU

nucleus. Our new system, founded on the screening of strains modified for sensitivity to toxic products, gives a very high percentage of incompatibility mutants in P. anserina. Compared with all mutants previously screened by other methods, the mutants described in this paper expressed new character- istics for the incompatibility spectra. It will be interesting to apply this method of selecting incompatibility genes in other fungi. This will allow one to verify whether vegetative incompatibility of other fungi is, as in P. anserina, related to plasma membrane proteins and differentiation.

We would like to thank MARTINE SABOURIN, EVELYNE ABELA and MARTINE VALENTIN for excellent technical assistance and J. M. BOVE for suggestions for the manuscript. This research was supported by the Conseil Scientifique de I’Universiti de Bordeaux 11, the Centre National de la Recherche Scientifique and the Mission de la Recherche (Aide i la Recherche Universitaire en Biologie).

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