Tke Use of Duplication-generating Rearrangements for Studying Heterokaryon Incompatibility Genes in Neurospora1

Heterokaryon (vegetative) incompatibility, governing the fusion of somatic hyphal filaments to form stable heterokaryons, is of interest because of its widespread occurrence in fungi and its bearing on cellular recognition. Conventional investigations of the genetic basis of heterokaryon incompatibility in N . crassa are difficult because in commonly used stocks differences are present a t several het loci, all with similar incompatibility phenotypes. This difficulty is overcome by using duplications (partial diploids) that are unlikely to contain more than one het locus. A phenotypically expressed incompatibility reaction occurs when unlike het alleles are present within the same somatic nucleus, and this parallels the heterokaryon incompatibility reaction that occurs when unlike alleles in different haploid nuclei are introduced into the same somatic hypha by mycelial fusion.-Nontandem duplications were used to confirm that the incompatibility reactions in heterokaryons and in duplications are alternate expressions of the same genes. This was demonstrated for three loci which had .previously been established by conventional heterokaryon tests-het-e, het-c and mt. These were each obtained in duplications as recombinant meiotic segregants from crosses heterozygous for duplicationgenerating chromosome rearrangements. The particular method of producing the duplications is irrelevant so long as the incompatibility alleles are heterozygous.-The duplication technique has made it possible to determine easily the het-e and het-c genotypes of numerous laboratory and wild strains of unknown constitution. In laboratory strains both loci are represented simply by two alleles. Analysis of het-c is more complicated in some wild strains, where differences have been demonstrated at one or more additional het loci within the duplication used and multiple allelism is also possible.-The results show that the duplication method can be used to identify and map additional vegetative incompatibility loci, without the necessity of heterokaryon tests. ENETIC factors other than mating type that prevent or impede the formaG tion of heterokaryons in Neurospora crassa were first described by GARNJOBST (1953, 1955) and by HOLLOWAY (1953, 1955). If two monokaryotic haploid strains differ in the alleles at one or more loci that govern heterokaryon incompatibility, stable heterokaryons are not formed between them. Four such incompatibility loci, het-c, -d, -e, and -i, have been identified using heterokaryons (GARNJOBST 1953, 1955; PITTENGER and BRAWNER 1961; WILSON and GARNJOBST 1966), and the mating-type alleles A and a act also as a pair of hetero1 Supported by Public Health Service Research Grant AI-01462 and Research Career Award K6-GM-4899. Genetics 80: 87-105 May, 1975. 88 D. D. PERKINS karyon incompatibility genes ( COONRADT 1943; BEADLE and COONRADT 1944; SANSOME 1946; GARNJOBST and WILSON 1956; PITTINGER 195 7; NEWMEYER, HOWE and GALEAZZI 1973). At least one other het-gene difference is known to be present in laboratory stocks (J. F. WILSON, personal communication). Cytological and chemical observations have been made on the heterokaryon incompatibility reactions (WILSON, GARNJOBST and TATUM 1961 ; WILLIAMS and WILSON 1966), whose behavior and specificity suggest that they may have a bearing on cellular recognition mechanisms in higher organisms. Heterokaryon or vegetative incompatibility independent of sexual compatibility is also found in other fungi such as Aspergillus (JINKS et al. 1966), Podospora (t and U genes-&ER and BLAICH 1973), and the slime molds (DEE 1966; COLLINS and CLARK 1968). The widespread occurrence of vegetative incompatibility differences suggests a significant selective role, the nature of which has been a subject of speculation (see JINKS et al. 1966; BUTCHER 1968; CATEN 1972; CLARK and COLLINS 1973; HARTI,, DEMPSTER and BROWN 1975). For reviews of heterokaryosis and its genetic control see DAVIS (1966) and CATEN and JINKS (1966). Because marker stocks and commonly used laboratory wild types of Neurospora crassa often differ at two or more het loci, a conventional genetic analysis is difficult, as with any multifactorial trait where the individual component genes cannot be distinguished phenotypically, Using the heterokaryon incompatibility associated with mating type, NEWMEYER (1965, 1970) circumvented the difficulty of analysis by constructing partial diploids, in which only one segment is present in heterozygous condition and the rest of the genome is haploid. Heterozygous partial diploids are readily obtained from crosses which involve chromosome rearrangements that generate nontandem duplications by meiotic recombination and chromosome segregation ( NEWMEYER and TAYLOR 1967; TURNER et al. 1969; PERKINS 1971, 1972a, 1974). Each such rearrangement produces predictably a class of progeny that are duplicated for a specific chromosome segment. If the segment includes a heterokaryon incompatibility locus, and if the two parents contribute different alleles at that locus, the incompatibility is manifested in each heterozygous offspring by a characteristic incompatibility phenotype. Thus, het loci can be identified and studied in one region at a time. Duplications have been used in this way by MYLYK (1975a) to identify at least five new vegetative incompatibility loci in N . crassa strains collected from nature. The usefulness of heterozygous nontandem duplications is not limited to studying incompatibility, and the approach used in this paper could equally well be applied to other situations where interactions are to be studied, as for example with regulatory genes (METZENBERG, GLEASON and LITTLEWOOD 1974). The main purpose of this paper is to show that the duplication method is valid and that incompatibility observed in duplications is in fact due to heterokaryon incompatibility genes. This has been accomplished for three of the loci previously known from their effects on heterokaryons-het-e (VIIL) , het-c (IIL) , and mating type (IL) -using appropriate rearrangements to produce INCOMPATIBILITY GENES IN DUPLICATIONS 89 duplications embracing each of them. Duplications covering het-e are reported here for the first time. Preliminary accounts were given earlier of het-c in duplications (PERKINS 1968, 1969, 1972b). Mating-type (A/a) heterozygosity has previously been studied using terminal duplications from a pericentric inversion (NEWMEYER and TAYLOR 1967) and interstitial duplications from an insertional translocation (PERKINS 1972a) ; these results are now extended using A l a duplications obtained in a third way which employs overlapping rearrangements whose breakpoints straddle the mating-type locus. MATERIALS A N D METHODS Strains: T(VII+IV)T54M50 originated in wild type 74A following UV in experiments of INOUE and ISHIKAWA (1970). T(II+V)NMl49 originated in wild type Em a following W in 1964 experiments of NOREEN E. MURRAY. T(1;V)ARIZ arose in a strain of mixed parentage following W in 1967 experiments of ALAN RADFORD. T(I;V)P5401 was found in a stock of his-4 (C141) received from BARBARA D. MALING in 1959. T(I;V)477lI arose from the cross Abb 4A X 25a following UV (BEADLE and TATUM 1945).T(ll+VI)P2869 was found in a cross of T(VI+[I;III]YI63329 X T(I;ll)P5390. All are phenotypically wild-type and have been separated from any mutant genes that may originally have been present. Standard wild types were 74-Om-1A and 74OR8-la or their derivatives OR23-1VA and ORSa (MYLYK, BARRY and GALEAZZI 1974). Standard testers for mating type (mt, A/a) and for aberration us. normal sequence were the isosequential fluffy strains f l P A and PPa. For information on the origin, characteristics and scoring of the markers used, see references given by BACHMANN and STRICKLAND (1965), BACHMANN (1970), or BARRATT and OGATA (1974). The meaning of locus symbols is given in the legend of Figure 1. Media and technical methods: Duplications heterozygous for different het alleles are initially abnormal in morphology and growth rate, and produce bro& pigment when grown on glycerol complete medium (GCP, TATUM et al. 1950) or on minimal medium (VOGEL 1964) with 1% sucrose plus L-phenylalanine, 0.2 mg/ml, and Ltyrosine, 0.5 mg/ml (PT) . On minimal medium without these amino acids the morphology is the same but no pigment is formed. Because the pigment is useful in scoring, ascospores were usually isolated to either GCP or PT medium. Crosses were carried out on synthetic crossing medium (SC) (WESTERGAARD and MITCHELL 1947) containing 2% agar and 2% sucrose, usually in 18 X 150 mm tubes. Mating type, fertility, and presence of an aberration or duplication were scored by fertilizing 5-day-old fluffy testers in individual 10 X 75 mm tubes of SC at 25" (TAYLOR 1965). Cultures were scored as aberrant when the ascospores shot from perithecia of these test crosses were about 75% black and 25% white (defective) for duplication-producing rearrangements, and 50%, 50% for reciprocal translocations; about 95% of ascospores are black in crosses between strains that are isosequential, either both Normal or both rearranged (PERKINS 1974). Methods of crossing, ascospore isolation, and stock preservation were as described previously (PERKINS 1959, 1962). RESULTS Three examples of heterokaryon (vegetative) incompatibility loci will be I. het-e testers and the manifestation of het-e in heterozygous duplications Construction, verification, and use of duplication-generating het-e testers: h t e , which was identified and mapped in VIIL by WILSON and GARNJOBST (1966), is contained in duplications generated by translocation T(VII+IV)T54M50, whose structure is shown in Figure 1A. A distal segment of VIIL given-het-e, het-c and mating type.