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Copyright 0 1986 by the Genetics Society of America CHARACTERIZATION OF THE HETEROKARYOTIC AND VEGETATIVE DIPLOID PHASES OF MAGNAPORTHE GRISEA MARK S. CRAWFORD,’ FORREST G. CHUMLEY: CAROLYN G. WEAVER BARBARA VALENT‘,’ AND Department of Chemistry, University of Colorado, Boulder, Colorado 80309 Manuscript received November 14, 1984 Revised copy accepted August 18, 1986 ABSTRACT The heterokaryotic and vegetative diploid phases of Magnaporthe grisea, a fungal pathogen of grasses, have been characterized. Prototrophic heterokaryons form when complementary auxotrophs are paired on minimal medium. Hyphal tip cells and conidia (vegetative spores) taken from these heterokaryons are auxotrophs with phenotypes identical to one or the other of the parents. M. grisea heterokaryons thus resemble those of other fungi that have completely septate hyphae with a single nucleus per cell. Heterokaryons have been utilized for complementation and dominance testing of mutations that affect nutritional characteristics of the fungus. Heterokaryons growing on minimal medium spontaneously give rise to fast-growing sectors that have the genetic properties expected of unstable heterozygous diploids. In fast-growing sectors, most hyphal tip cells are unstable prototrophs. The conidia collected from fast-growing sectors include stable and unstable prototrophs, as well as auxotrophs that exhibit a wide range of phenotypes, including many recombinant classes. Genetic linkage in meiosis has been detected between two auxotrophic mutations that recombine in vegetatively growing unstable diploids. The appearance of recombinants suggests that homologous recombination occurs during vegetative growth of M. grisea. No interstrain barriers to heterokaryosis and diploid formation have been detected. The mating type of the strains that are paired does not influence the formation of heterokaryons or diploids. M AGNAPORTHE grisea is the name given to the perfect (sexual) state of what was formerly considered to be two fungal species: Pyricularia oryzae, pathogens of rice (Oryza sativa), and Pyricularia grisea, pathogens of grasses other than rice (BARR1977; YAEGASHIand UDAGAWA 1978a,b). Rice-infecting strains are the agents of rice blast disease, which causes serious damage to crops in many areas of the world. Whereas M . grisea as a species has a very broad host range, any particular isolate is able to infect only one or a few ’ Present address: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99163. * Present address: Central Research and Development Department, The DuPont Company, Experimental Station, E402/2208, Wilmington, Delaware 19898. Author to whom inquiries should be directed. ’ Genetics 1 1 4 1 1 1 1 - 1 129 December, 1986. 1112 M. S. CRAWFORD E T AL. species of grasses (ASUYAMA1965; KATO 1978). Hundreds of races are distinguished among rice-infecting strains according to their ability to infect particular cultivars of rice (LATTERELL,MARCHETTI and GROVE1965; ATKINSet al. 1967; KIYOSAWA1976; YAMADAet al. 1976; Ou 1980). M . grisea thus offers a unique opportunity to study the genetic basis of host species and cultivar specificity in a fungal plant pathogen. Although pathogenic variants and auxotrophic mutants of M. grisea have been reported (YAEGASHI1978; YAEGASHI and ASAGA1981; TAGA et al. 1982; NAGAKUBO et al. 1983), this important pathogen is still undeveloped as a subject for rigorous genetic analysis. The work reported here was undertaken to characterize heterokaryons and diploids of M . grisea in order to utilize these forms of the fungus in conducting genetic analysis. Either a heterokaryotic or a diploid form of the fungus is necessary for complementation and dominance testing of genetic traits of M. grisea, which grows normally as a haploid. This study presents new results in several areas. We have discovered that complementary M. grisea auxotrophs form prototrophic heterokaryons in which conidia and single hyphal tip subcultures are auxotrophs with phenotypes identical to one or the other of the parental strains, as first described for the plant pathogen Verticillium dahliae (PUHALLAand MAYFIELD1974). Fast-growing sectors that emerge from heterokaryons are at least transiently diploid. These diploid cultures of M. grisea are highly unstable; conidia derived from them include stable prototrophs (which are probably haploid) and auxotrophs, including many that have undergone recombination of markers in the parents. This observation confirms previous reports (YAMASAKI and NIIZEKI 1965; GENOVESIand MAGILL1976) that the parasexual cycle (PONTECORVO 1956) is active in M . grisea. Conidia that show properties expected of unstable diploids have also been detected, although these are rare among the conidia obtained from diploid mycelia. T w o auxotrophic mutations that are linked meiotically recombine in the parasexual cycle, suggesting the occurrence of intrachromosomal mitotic recombination in M. grisea. We have determined that the mating-type locus in M. grisea does not condition vegetative incompatibility as does the mating-type locus in Neurospora crassa (BEADLEand COONRADT 1944). Finally, no interstrain barriers to the formation of heterokaryons have been detected in our studies. MATERIALS AND METHODS The organism: M. grisea is a filamentous heterothallic Ascomycete, group Pyrenomycetes. It is the perfect state of the plant pathogens, P. oryzae (pathogens of rice), and P. grisea (pathogens of grasses other than rice). No morphological distinction can be made between the two groups of pathogens. In a number of laboratories, including our own, rice-infecting strains have been crossed with strains that infect other grasses (TANAKA, MURATAand KATO 1979; YAEGASHI and ASAGA1981; VALENTet al. 1986; M. TAGA,personal communication). Progeny from these crosses can be backcrossed to both parents, yielding viable progeny (VALENT et al. 1986). Since no basis exists for the species distinction between strains that infect rice and strains that infect other grasses, we now refer to all Pyricularia strains as M. grisea. The asexual spores of M. grisea initiate infections on leaves, nodes and panicles of susceptible grasses. A single conidium contains three cells, each with a single nucleus. M . GRISEA HETEROKARYONS AND DIPLOIDS 1113 All three nuclei have a common origin and, thus, are genetically identical (YAMASAKI and NIIZEKI 1965). On the host plant, germ tubes emerge from one or more of the cells of a conidium and penetrate directly through the cuticle by means of an appressorium. Hyphae grow intracellularly within the host tissue. Conidiating lesions occur 57 days after initiation of infection. Resistance to this pathogen in plant tissue is often associated with a hypersensitive response, the death of the first host cells to come in contact with fungal hyphae (SUZUKI 1965). M . grisea is normally isolated as a haploid, with six chromosomes per nucleus (YAEGASHI and HEBERT1976; TANAKA, MURATAand KATO 1979; LEUNC1984). The hyphae are septate containing one nucleus per cell. Strains: The M.grisea field isolates used to develop the strains described in this study were SM81-11, SM81-4, WGG-FA40, Ken60-19 (all generously provided by H. YAEGASHI) and 0-42 (collected by H. KATO and B. VALENT,Tochigi Prefecture, Japan). These isolates originated from lesions either on finger millet (Eleusine coracana) or rice (Oryza sativa) growing in Japan. Specific strains used in this study are listed in Table 1 . Most of the auxotrophic mutants studied were isolated from parental strains several generations removed from the original field isolates. Several multiply marked strains were used to form heterokaryotic and diploid colonies. [Unless otherwise indicated, the data listed in Tables 5, 6 and 7 are derived from pooling the results of experiments that involved several closely related strains that contain the same markers (see table legends)]. Most of the strains given a number preceded by the letters “CP” were isolated following mutagenesis. Strains designated by a three-part number are single ascospore progeny of genetic crosses. The first part of the strain number indicates the cross serial number, the second is the ascus number and the third is the ascospore number. Formerly, the two mating types of M. grisea were referred to as “A” and “a” (KATO, YAMACUCHIand NISHIHARA1976; YAEGASHIand NISHIHARA1976). In order to use a consistent nomenclature at all loci and to avoid implying a dominance relationship between these alleles, we refer to “A” as m a t l - l and “a” as matl-2. We have followed the conventions for genetic nomenclature suggested by YODER,VALENTand CHUMLEY (1 986). A three-letter gene designation that recalls the relevant phenotype has been assigned to all mutations ( i e . , lys). A permanent, unique, serial allele number has been assigned to each mutation, and it follows the gene designation, separated by a hyphen ( i . e . , lys-1). In many cases, mutations at more than one genetic locus can lead to the same phenotype. Where such loci have been distinguished through complementation and recombination tests, a locus number has been assigned. The locus number follows the gene designation immediately, preceding the allele number, which is still set off by a hyphen (i.e., lysl-1). All cultures are stored dried and frozen in cellulose filter paper, et al. 1986). in silica gel or in infected host tissue (VALENT Media: All media used in this study were solidified with 1.5%agar unless otherwise stated. Minimal: 1 % sucrose, Vogel’s N Salts (VOGEL1964), 1 mg/liter thiamine, 5 pg/liter biotin. Minimal plus sorbose: 0.15% sucrose, 3% sorbose, Vogel’s N Salts, 1 mg/liter thiamine, 5 pg/liter biotin. Complete: 1 % sucrose, 0.6% yeast extract, 0.6% casein enzymatic hydrolysate. Complete plus sorbose: 0.05% glucose, 0.05% fructose, 3% sorbose, 0.6% yeast extract, 0.6% casein enzymatic hydrolysate. Misato-Hara medium: 1 % soluble starch, 0.2% yeast extract (TAGA et al. 1982). Oatmeal: 50 g of rolled oats are heated in 500 ml of water at 70” for 1 hr, then are filtered through cheesecloth. The volume of the filtrate is adjusted to 1 liter with water. Mutagenesis and detection of auxotrophs by replica printing: Conidia are washed from a culture growing on oatmeal agar and diluted to 1 X 106/ml in sterile 0.025% Tween 20. Five ml of this spore suspension are stirred in a 3.5-cm Petri dish while 1114 M. S. CRAWFORD ET AL. TABLE 1 M. grisea strains Strain Mating type Ken6O- 19 0-42 SM81-11 matl-2 matl-1 matl-1 SM81-4 matl-2 WGG-FA40 matl-1 CP8 CP18 CP20 CP21 CP22 CP28 CP3 1 CP62 CP103 CP125 CP141 CP143 CP167 70-5-7 281-9-2 859-15-4 1951-24-2 3596-5-1 3829- 1-2 3829-1-6 3995-26-1 3995-26-4 3998-4-3 4007-9-1 4007-17-2 4065-5-2 4065-1 1-1 4065-20-1 4069-4-2 4069-5-1 4069-5-4 4069-8-1 4069-8-3 4069-1 1 - 1 4069-1 1-2 4075-7-2 4075-29-1 4085-3-4 4085-9-2 4086-2-2 4089- I - 1 matl-1 matl-1 matl-1 matl-1 matl-1 matl-1 matl-l matl-1 matl-1 matl-1 matl-1 mall-2 matl-2 mall-I matl-1 matl-2 mall-1 matl-1 matl-2 matl-1 - matl-1 mat 1-2 matl-2 matl-2 matl-1 matl-2 matl-1 matl-1 matl-1 matl-1 - matl-2 matl-2 matl-2 matl-1 matl-2 matl-2 - Genotype Rice pathogenic field isolate (H. YAEGASHI) Rice pathogenic field isolate (B. VALENTand H. KATO) Finger millet and goosegrass pathogenic field isolate (H. YAEGASHI) Finger millet and goosegrass pathogenic field isolate (H. YAECASHI) Finger millet and goosegrass pathogenic field isolate (H. YAEGASHI) arg-I ade-8 lys-1 lys-2 lys-3 phe-I ser-2 buf-4 sor-1 I arg-6 asn-2 Rice pathogenic laboratory strain nic-2 Prototrophic laboratory strain Prototrophic laboratory strain Goosegrass pathogenic laboratory strain Goosegrass pathogenic laboratory strain Prototrophic laboratory strain arg-1 buf-4 arg-1 buf-4 buf-4 phe-1 arg-1 phe-1 lys-2 arg-1 lys-1 arg-1 buf-4 lys-I arg-I ser-2 ser-2 arg-1 ser-2 arg-1 phe-1 arg-1 phe-I arg-1 phe-1 arg-l phe-1 arg-1 phe-1 arg-1 phe-1 arg-1 phe-1 arg-1 lys-I ser-2 lys-I ser-2 phe-1 sor-11 buf-4 phe-1 sor-1 I ade-8 buf-4 phe-I . . arg-1 ser-2 M . GRZSEA HETEROKARYONS AND DIPLOIDS 1115 TABLE 1-Continued ~ ~ Strain ~~ Mating type 4089-2-1 4089-2-4 4089-4-1 4089-8-1 4089-8-5 4089-9-3 4089-10-2 4089-1 2-2 4091-5-8 matl-1 matl-2 matl-2 4 105-1-1 4105-1-2 41 05-1-3 matl-2 matl-I matl-2 mall-2 - Genotype ser-2 ade-8 buf-4 ser-2 ade-8 buf-4 ade-8 buf-4 ser-2 arg-I bu.4 ser-2 ade-8 buf-4 ade-8 buf-4 ser-2 ade-8 buf-4 Goosegrass and weeping lovegrass pathogenic laboratory strain phe-1 ser-2 sor-1 I phe-1 ser-2 sor-I1 are-I ser-2 sor-I 1 Strains of M. grisea used in this study. The phenotypes conferred by mutations are listed in Table 2. The two mating-type alleles are matl-1 and matl-2. For some strains, mating type has not been determined (indicated in the table as -). being irradiated with UV light for 4 min (Ultraviolet Products Mineralight, Model No. UVG-11, placed 9 cm above the conidial suspension). This treatment kills 93-98% of the conidia. The irradiated conidia are diluted and spread on complete plus sorbose medium. Sorbose inhibits radial growth of M . grisea, causing the formation of compact colonies and allowing up to 200 viable conidia to be plated per dish. After 24 hr, a piece of sterile cellulose filter paper is placed over the surface of the plate, and the colonies are allowed to grow into the filter paper for 2 days at room temperature. The filter paper is then transferred to minimal plus sorbose medium for replica printing. After 2 days, the prototrophic colonies have grown into the minimal medium and the filter paper is discarded. Comparison of the replica plates reveals the auxotrophic colonies, which are picked off the complete plus sorbose plate and are tested to determine the nature of the auxotrophy. All putative auxotrophs are stored immediately in dried, frozen filter paper (VALENTet al. 1986). Heterokaryon and diploid formation: When two complementary auxotrophs are paired on minimal medium, prototrophic heterokaryotic growth appears 1-3 wk after pairing. No growth occurs when the two strains are separated by a dialysis membrane. Fast-growing sectors arise spontaneously when these heterokaryons are grown on minimal medium (within 1-3 wk at 25"). The fast-growing sectors have the properties of unstable diploids, as described below. A faster method for obtaining heterokaryotic growth involves cocultivating auxotrophic strains on complete medium. After 3 days, cocultivated mycelia are transferred to minimal medium. Prototrophic growth appears within 2 days. Prototrophic growth from paired auxotrophs is initially two to three times slower than wild-type growth, and the colonies are very compact, similar to wild-type growth on sorbose-containing media. Genetic crosses: Sexual crosses of prototrophic strains are performed by pairing strains of opposite mating type on oatmeal agar and allowing the two strains to grow together at 21" under fluorescent light. Perithecia form at the intersection of growth between the two strains. Sexual crosses between an auxotroph and a prototroph or between two noncomplementary auxotrophic strains are performed by placing plugs from the strains to be mated on Misato-Hara medium and incubating them as above. Crosses between complementary auxotrophic strains of opposite mating type are accomplished by forming a heterokaryon between the two strains, which is then transferred to oatmeal medium and incubated as above. Perithecia appear 2-wk later. Tetrads are 1116 M. S . CRAWFORD E T AL. dissected by hand on 4% water agar (supplemented with the nutrient requirements of the parental strains) using a finely drawn glass needle and a 50X Wild stereomicroscope. Germinated ascospores are transferred from the Supplemented water agar to complete medium supplemented with the nutrient requirements of the parental strains. Cytology: T h e number of nuclei per cell in conidia was determined by staining with 4 ',6-diamidino-2-phenylindole (DAPI) (RUSSELL, NEWMAN and WILLIAMSON 1975). Conidia were collected by washing a culture growing on solid medium with trichloroethane. T h e spore suspension was evaporated to dryness, and spores were resuspended in 3 ml of H 2 0 containing 3 mg of DAPI, 100 mM Tris, 100 mM NaCI, 10 mM EDTA, 0.01% (v/v) Triton X-100, pH 7.0. Nuclei were visualized using incident light from a mercury lamp. A filter with a high wavelength cutoff at 360 nm was placed between the light source and the sample, and another filter with a low wavelength cutoff at 436 nm was placed in the tube of the microscope. Stained nuclei fluoresce bright green. Cross walls in conidia are easily visualized using illumination from a tungsten lamp. RESULTS Isolation of mutants: Strains that showed either interesting pathogenicity traits or better than average sexual fertility were chosen for mutagenesis. Twenty-one auxotrophs were isolated from three rice-infecting strains. Two of these rice-infecting strains were field isolates (Ken6O-19) and 0-42), and the third (CP143) was a single ascospore progeny of a cross between Ken60-19 and 3596-5-1, a strain developed in our laboratory. The remainder of the auxotrophs were isolated from finger millet-infecting isolates (SM81-11 and WGG-FA40), from progeny of crosses between different finger millet-infecting isolates (281-9-2 and 70-5-7) and from a strain derived from a cross between a finger millet-infecting isolate and a weeping lovegrass-infecting isolate (409 15-8). The mutants recovered are shown in Table 2. The yield of auxotrophic colonies following UV mutagenesis (approximately 0.35% of the survivors) is consistent with each of the mutagenized strains being haploid. Colonies with growth that is not restricted by sorbose (Sor-) arise at a frequency of about Colonies with a particular defect in pigmentation (Buf-) arise at a frequency of about to Meiotic analysis of M. grisea mutants: Auxotrophic, sorbose-resistant and pigmentation-defective mutants were crossed with wild-type strains. Table 3 shows the results of both random ascospore and tetrad analysis including all asci in which five or more ascospores germinated. Ascospore germination in these crosses ranged from 20 to 80% depending on the strains involved. In all cases, segregation in tetrads is consistent with a single-gene defect causing the phenotype. No aberrant ratios within a tetrad were seen. Mutant strains were intercrossed to construct multiply marked strains and to determine linkage relationships. Results of both random ascospore and tetrad analysis are shown in Table 4. All markers appear unlinked, with the exception of ade-8 and phe-1, which are separated by a distance of 8.3 map units. No tetrads were seen that would indicate that any phenotype is the result of more than one mutation. The segregation patterns presented in Tables 3 and 4 are entirely consistent with M . grisea being haploid. Heterokaryons: We have observed prototrophic growth resulting from the pairing of many different complementary auxotrophs, examples of which are M . GRZSEA HETEROKARYONS AND DIPLOIDS 1117 TABLE 2 Mutant strains of M. grisea Genotype adeahsargamatz buf CYh cys- g1uhishomi1vinoleu1ys- metnit$heserSOT tyrVal- No. of isolates 17 2 10 5 1 76 60 4 1 4 2 3 2 4 6 16 3 1 5 18 1 1 Phenotype Requires adenine Requires adenine and histidine Requires arginine Requires asparagine Aminotriazole resistance Pigmentation block Mycelium buff instead of gray Cycloheximide resistance Requires cysteine or methionine Requires glutamate Requires histidine Requires homoserine or methionine and threonine Requires isoleucine and valine Requires inositol Requires leucine Requires lysine Requires methionine Requires nicotinamide Requires phenylalanine Requires serine Sorbose resistance Requires tyrosine Requires valine Auxotrophic, drug-resistant and morphological mutants derived by UV mutagenesis. Some of the buf - mutants indicated appeared spontaneously. shown in Table 5 . Such prototrophic growth could result from any one of several factors: one or both of the auxotrophic strains could have reverted to prototrophy, the paired strains could be cross-feeding, the colony could be diploid or the colony could be heterokaryotic. The use of double auxotrophs in these studies reduces the chances that prototrophic growth is due to reversion of auxotrophic mutations. Reversion to prototrophy is ruled out by the recovery of all auxotrophic markers originally present in the parental strains (Table 5 and Table 6). The fact that prototrophic growth does not occur if the parental strains are separated by a dialysis membrane indicates that the prototrophic growth is not due to cross-feeding. If prototrophic growth were the result of the formation of stable heterozygous diploid nuclei, then conidia harvested from the colony would be expected to be diploid and prototrophic. However, the prototrophic growth gives rise only to auxotrophic conidia (Table 5). If prototrophic growth were due to the formation, breakdown and continued reformation of unstable heterozygous diploid nuclei, then some of the conidia recovered should exhibit recombination of the parental markers due to mitotic crossing over and/or chromosome loss during reversion to haploidy (parasexual recombination). N o such recombinants are detected (Table 5). Thus, the prototrophic growth resulting from the pairing of two M. grisea 1118 M. S. CRAWFORD E T AL. TABLE 3 Segregation ratios of alleles at loci controlling morphology and nutritional characteristics No. of tetrads recovered Ratio in a tetrad ade-8 buf-4 lys-I 1:4 2:3 3:2 4: 1 2:4 3:3 4:2 3:4 4:3 4:4 Aberrant 1 1 2 1 3 2 1 1 2 1 6 5 1 1 7 4 11 17 0 1 2 0 0 2 2 2 1 4 3 1 0 55 7 5 17 22:27 21:18 154:118 Total no. of tetrads Random ascospores 6 3 1 0 21 110:111 193:225 lys-2 2 1 1 1 1 2 phe-I 1 1 ser-2 1 2 2 1 4 2 3 5 6 sor-11 arg-l 4 2 1 6 3 1 1 10 7 9 0 1 1 1 0 5 5 35 10 41 175:185 81:105 3 0 420:332 The data presented are the pooled results of a large number of crosses in which strains that carried the mutant allele indicated were mated with strains that carried the wild-type allele. The strains that were crossed are included in Table 1 . The column headed “Ratio in a tetrad” reports the results from tetrads in which five or more of the eight ascospores germinated. The ratio of wild-type to mutant ascospores is presented. Results from random ascospore analysis are given as the ratio of wild-type to mutant ascospores. The segregation of mating type has been observed among the progeny of hundreds of crosses; the ratio of the two mating types is always 1 : l . strains carrying complementary auxotrophies is not due to reversion, crossfeeding o r heterozygous diploidy and must be due to heterokaryosis. M. grisea heterokaryons can be propagated by mass hyphal transfers taken behind the leading edge of the colony. However, single hyphal tips from the leading edge of the heterokaryon are auxotrophic and carry all markers corresponding to one or the other of the parental strains (Table 6). The conidia derived from heterokaryons are also auxotrophic and are identical to one or the other of the parental strains (Table 5 ) . Neither prototrophic conidia nor conidia that are recombinant for the auxotrophies present in the parents have been recovered from heterokaryons. Diploids: Fast-growing sectors spontaneously emerge from heterokaryons after 1- to 3-weeks’ growth on minimal medium (Figure 1). These sectors have a growth rate similar to wild type, but are morphologically distinct, with a very sparse appearance. In contrast to hyphal tip cultures from heterokaryons, most hyphal tip subcultures from fast-growing sectors are prototrophic. Conidia taken from the fast-growing sectors show a wide range of phenotypes, including classes that exhibit recombination of the parental markers (Table 7). This observation provides evidence that fast-growing sectors arising from heterokaryotic cultures are at least transiently diploid. These experiments were conducted by first purifying prospective diploids through single proto- 1119 M. GRISEA HETEROKARYONS AND DIPLOIDS TABLE 4 Recombination frequencies between several loci that control morphological and nutritional characteristics Second marker First marker arg-1 lys- 1 phe-1 1461261 1:5:9 1331260 5:2:12 sor-1 I ser-2 lys- 1 18/50 0:0:4 #he-1 bu.4 1471298 3:5:19 931135 2:3:10 521110 3:1:5 9/39 1:0:4 44/86 931151 1:2:10 sor-I1 24/42 0:0:4 ser-2 15/33 2:2:17 ade-8 45/98 1:l:S 8/ 109 16:l:l 45/98 1:2:8 The data presented are the pooled results of a large number of crosses in which strains that carried the mutations indicated were mated as described in the text. T h e strains that were crossed are included in Table 1. The fractional number is the fraction of the total progeny examined by random ascospore analysis that were recombinant for two traits involved. The three-part ratio is parental ditype:nonparental ditype:tetratype tetrads. TABLE 5 Nutritional requirements of conidia harvested from heterokaryotic colonies Strains paired ade-8 ser-2/arg-1 phe-1 arg-1 phe-1 /lys- 1 ser-2 arg-1 #he-llade-8 buf-4 ser-2 arg-1 ser-2 sor-1 l l a d e - 8 buf-4 Frequency of recovered phenotypes 1918/3162 Ade- Ser18/40 Arg- Phe591274 Arg- Phe221134 Arg- Ser- Sor- 1244/3162 Arg- Phe22/40 Lys- Ser2151274 Ade- Buf- Ser1121134 Ade- Buf- Strains carrying complementary auxotrophic mutations were paired on complete medium and allowed to grow together for 2 days. The cocultivated mycelium was then transferred to minimal medium. Prototrophic growth emerged 2-5 days later. Conidia were harvested from this prototrophic growth, and their nutrient requirements were determined. In this table the ade-8 buf4 ser-2 strains are 4089-2-4, 4089-8-1 and 4089-10-2. These same strains were used for the ade-8 ser-2 pairing listed. Buf- was not scored among conidial colonies for this pairing. The arg-1 #he1 strains are 4069-4-2, 4069-1 1-1 and 4069-1 1-2; the lys-1 ser-2 strain is 4075-29-1; the ade-8 buf4 strain is 4089-4-1; and the arg-1 ser-2 sor-11 strain is 4105-1-3. trophic hyphal tips taken from fast-growing sectors. T h e hyphal tip cultures were grown on oatmeal agar, and conidia from these cultures were collected and plated on complete plus sorbose medium. Auxotrophs were identified by replica printing. T h e phenotypes of colonies obtained in two different sets of experiments are shown in Table 7. Monoconidial colonies from fast-growing sectors of arg-1 phe-lllys-1 ser-2 heterokaryons include many recombinant classes. Every possible recombinant class except Arg- Ser- and Arg- Lys- Seris represented. T h e nonrecombinants (Arg- Phe- and Lys- Ser-) are not the major classes. Similarly, monoconidial colonies derived from a fast-growing sector that emerged from a heterokaryon formed between an arg-1 phe-1 par- P E : 2 $4 E2 n 2. <S? 1 rb n e, 0 7 5.m 3 - y s o ra v, 2.09 3 "2 ah;, (r, r 4 0 9 % w -<r c d o4 L m ri; ' I g.s ra - T ; ; . i r 2 xnra 3z &U - 3 !U w -0 = $4 1 $7 ' a % $g 2 s $ om B % ra r *2 0 'p 2 2 T.3.2 w . g 5 2 m & z 2 z.c zi 2 % SZZ? 22ra ra e, T m Y.-ra P o = 2 . 3 5 gab = 1 4 2 $ m c 2 3 2 5 . a n 4 "%s 0 3 m raTs;$ 3 g.- 0 * 5 0 T? 3 2 5*:.0r) % 3 e, % Q 0 0-h 7 P% 2 ?rt $4 5 2 2; 2d - 0 4 g -.a n 5 3 !U= E ? --.e3 s 05 -s g:> w $8 w w). 0 0 s L? 2. *.q + V I ? 0 ?:!ai? ,& z m m E ? a 2.q $ ; 7'0.0;: J 2 g k 0s 2. -2 5. ' S S 7 h -% > $ E 0 3 2.0: g * g , " raxp Oz= <sragc ra2 0 0 g 3 geDap8 $ 8 g z . q 3 @ ?3 r 0 , S W % C $ mS.E.3 g3 L ar;:":$ a s ? g*g 2 s:2sgss2 p ; a- C O 4 ;;.%:% 2-7 2n 4e, at 2 x 3 $ 2 S y ; ; . 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Y5' 3 E S N La F> ? 4 . w -5.09, U I w w 'Plp+*w 3 -4 0 r r m w n 9 Lrq 7 O, -ps&%=- m c -;zg: &w n * 7 OJ *,'t*fi 5 557 LkL'U w k ~ ' Z c ~ ggNn FE!?*' 2. e, V I , % a " q J O - 3 d L CI m> v, c) '3: rQ0 i 5 2 e. y 8 b 42 2 5 r p e m p r h Y b u g 5 ; : % g. 29 e F! 7. $ >> z 3. 2. 5g2$;;& =?I 2 s' z 'w; c G g a o % $0.52 - = I2. g o u c ?.? ' I U Y V &"Lp - 5 : k2 fidoco. 't a n* wwgmc~$g 2: % *yg -c z3 22 n P F v) Lags', 3 M . GRZSEA HETEROKARYONS AND DIPLOIDS 1121 TABLE 7 Nutritional requirements of conidia harvested from purified diploid colonies Strains paired arg-I phe-lllys-I ser-2 Frequency of recovered phenotypes 1347 + 1 Arg- Phe- 5 10 125 3 149 1 3 47 1 1 2 2 Lys- SerArgPheLysSerArg- LysLys- PhePhe- SerArg- PheArg- LysLys- PheArg- Lys- SerPheSerPhe- Ser- 1697 Total tested arg-1 phe-llade-8 buf-4 ser-2 17 8 15 1 1 23 3 1 1 + AdeBufSerAdeAdeBufPheBuf- Buf - SerSerBufPhe- Ser- 70 Total tested Heterokaryons were formed by pairing strains that carried the mutations shown. From these heterokaryons, fast-growing sectors arose spontaneously. From each fast sector, a single hyphal tip was transferred to minimal medium and incubated for several days. The colony growing on minimal medium was subcultured to oatmeal medium by mass hyphal transfer. After 2 weeks' incubation at 25", conidia were harvested from the oatmeal plates and plated on complete plus sorbose medium. The resulting colonies were replica-printed to minimal plus sorbose medium to identify auxotrophs. The nutritional requirements of the auxotrophs were then determined. The 1697 conidia tested in the first part of the table (derived from arg-1 phe-1 and lys-I ser-2 pairings) represent data pooled from six independent experiments that involved strain 4075-29-1 as the lys-1 ser-2 parent and strains 3995-26-4, 4069-5-1, 4069-5-4, 4069-8-1, 4069-11-1 or 4069-11-2 as the arg-I phe-1 parent. Note that these arg-I phe-I strains represent both mating types. The 70 conidia tested in the second part of the table represent the data from a single experiment involving strains 4069-1 1-2 (arg-I phe-I) and 4089-8-1 (ade-8 buf-4 ser-2) as parents. The conidia from diploid cultures that give rise to prototrophic colonies could be either heterokaryotic, diploid or haploid recombinants generated by the parasexual cycle. Over 2000 conidia from diploid cultures were examined using DAPI staining to visualize nuclei. All conidia contained only one nucleus per cell. Since the three nuclei in a conidium are genetically identical due to 1122 M. S. CRAWFORD E T AL. their common origin, germ tubes originating from different cells within the same conidium could not form prototrophic heterokaryons. Prototrophic colonies arising from recombinant haploid conidia would be expected to yield only prototrophic conidia, whereas colonies arising from heterozygous diploid conidia should yield some auxotrophic conidia through mitotic crossing over and/or haploidization. Thirty prototrophic colonies that arose from single conidia isolated from the fast-growing sectors shown in Table 7 were cultured on oatmeal medium. Conidia were harvested from each of these prototrophic cultures. The 3000-4000 conidia derived from each of the 30 prototrophic colonies were all prototrophic. Thus, the 30 colonies contained either haploid prototrophic or stable diploid mycelia. The latter possibility seems unlikely since the original diploid culture yielded auxotrophic conidia at a high frequency, indicating that in M. grisea the vegetative diploid stage is inherently unstable. The conclusion that these prototrophic strains are haploid is supported by the observation that they are stable to treatment with p-fluorophenylalanine o r UV-light, treatments that induce haploidization or homozygosis in diploids (LHOAS1961; WOODand KAFER 1969). Isolation of diploid conidia: Numerous attempts to isolate diploid cultures by collecting conidia from diploid mycelia and plating them on minimal medium have failed to reveal any unstable (heterozygous) prototrophs. This is consistent with the following visual observations of diploid cultures (Figure 2). Diploid cultures derived from single prototrophic hyphal tips produce sparse, rapidly spreading growth with very few conidia. One-wk-old mycelium from these cultures begins to papillate; that is, islands of heavy growth appear, some of which conidiate heavily. On minimal medium, stable prototrophs, which are presumably haploid breakdown products, appear and quickly overgrow the diploid culture. On complete medium (Figure 2), the papillae include recombinant auxotrophic hyphae as well as prototrophic hyphae. Some papillae from the diploid in Figure 2 carry the arg-l mutation as well as various combinations of phe-1, ade-8, ser-2 and buf-4. This is in contrast to the result described in Table 7 with the same diploid in which no conidia carrying the arg-l mutation were recovered. This supports the idea that arg-1 was not recovered among conidia produced by this diploid, due to the poor conidiation of hyphae carrying the arg-l mutation. Unstable prototrophic conidia that are presumably diploid were isolated following careful visual inspection of mycelium that had not yet begun to papillate. Such mycelium contains very few conidiophores; these are elongated in comparison to normal haploid conidiophores and contain only one or two relatively large conidia. Conidia picked from these conidiophores, using a fine platinum wire loop full of sterile 0.025% Tween 20 solution, produce colonies that subsequently give rise to conidia with recombinant phenotypes, as shown in Table 8. Thus, these rare conidia show properties expected of unstable diploids. Detection of intrachromosomal mitotic recombination: The arg-1 phe-l/ ade-8 buf-4 ser-2 putative diploid cultures described in Tables 7 and 8 were formed from parents with the same mating type. Pairing of these strains has M . GRISEA HETEROKARYONS AND DIPLOIDS 1123 FIGURE2.-Papillation in a diploid culture. A single hyphal tip was taken from a fast-growing sector that emerged from a heterokaryon formed on minimal medium between strains 4069-1 1-2 ( m a l l - 2 arg-l $he-1) and 4089-8-1 ( m a l l - 2 ade-8 buf-4 ser-2). This tip was subcultured on complete medium. T h e figure shows this subculture after 14 days’ incubation at room temperature. Note the sparse, rapidly spreading hyphae (Hy) at the leading edge of the colony and the papillae (P) that have appeared in the interior. Both Buf- and normally pigmented (Buf+) papillae occur in approximately equal numbers. T h e significance of these papillae is discussed in the text. T h e bar is I cm in length. never produced perithecia, the organs of the sexual cycle. T h e putative diploids contain a pair of auxotrophic mutations that show linkage in meiosis, ade8 and phe-Z (Table 4). Since ade-8 and phe-1 were introduced in different parents, a reciprocal recombination event between the loci would generate one homologue with both prototrophic alleles, whereas the other homologue would carry both the ade-8 and phe-Z mutations. Such a mitotic recombination event, followed by haploidization, would yield prototrophic haploids and haploids with both auxotrophic markers. Stable prototrophic progeny have been recovered from all experiments with the arg-Z phe-Zlade-8 buf-4 ser-2 diploids. However, the frequency with which stable prototrophs are observed is reduced in comparison to experiments involving the arg-Z phe-ZlZys-Z ser-2 diploids (Table 7). T h e higher frequency of stable prototrophs recovered from the arg-Z phe-Z/ Zys-Z ser-2 diploids may be due to an arrangement of these markers that does not require an intrachromosomal mitotic recombination event for recovery of prototrophic haploids, but merely depends on independent assortment of chromosomes. Progeny containing both ade-8 and phe-Z have been recovered from diploids 1, 3 and 4 in Table 8. These prototrophic and double auxotrophic 1124 M. S. CRAWFORD ET AL. TABLE 8 Spontaneous haploidization in diploid cultures derived from single conidia Colonies identified from Phenotypes recovered BufAdeBufAdeAdeBufPheAdeAdeBufPheAde- + SerBuf- SerBuf- Phe- SerBufPhe- Ser- Monoconidial Monoconidial Monoconidial Monoconidial 1 2 3 4 1 2 1 1 1 14 6 14 SerPheSerPhe- 2 2 2 2 3 0 0 5 5 0 1 0 8 16 13 7 14 2 1 1 0 0 0 1 1 5 3 1 4 3 1 2 0 4 10 9 1 0 1 0 2 2 2 3 The nutritional requirements of conidia harvested from four independently cultured prospective diploid conidia derived from pairing 4089-8-1 (matl-2 ade-8 bu.4 rer-2) and 4069-1 1-2 (matl-2 arg-I p h e - l ) are shown. These prospective diploid conidia were isolated by picking single conidia from distinctive conidiophores produced by diploid mycelium, using a fine platinum wire loop holding a drop of sterile 0.025% Tween 20 solution. The data were obtained as described in the legend to Table 7. recombinants suggest that homologous recombination occurs during the vegetative growth of M. grisea diploids. Normally pigmented gray prototrophic colonies were at least ten times more abundant than Buf- prototrophic colonies among the recombinants derived from the arg-1 phe-llade-8 buf-4 ser-2 diploid cultures (Tables 7 and 8). In some experiments only gray prototrophs were recovered, even though auxotrophs with the B u f phenotype were recovered in high frequency. This asymmetry suggested to us that the buf-4 allele might be carried on the same chromosome as one of the auxotrophic markers in the Buf- parental strain. Indeed, data from five independent experiments (Tables 7 and 8) show that from 77 to 93% of the buf-4 colonies recovered also have the ser-2 mutation. Thus, these data suggest that buf-4 and ser-2 are located on the same chromosome, and occasional recombinants may be due to mitotic crossing over. Since the two mutations are not linked meiotically (Table 4), the proof that they reside on the same chromosome must await further experimentation. Vegetative incompatibility and complementation: No interstrain barriers to the formation of heterokaryons or diploids have been detected. Although some of the auxotrophic strains that could be expected to complement do not, there is as much apparent incompatibility between auxotrophs derived from a common parent as there is between auxotrophs derived from different parents (Table 9). The mating type of the paired strains has no effect on heterokaryon or diploid formation. The mutants carrying lys-I, lys-2 and lys-3 were independently isolated. Based 1125 M . GRISEA HETEROKARYONS AND DIPLOIDS TABLE 9 Complementation of auxotrophic mutations Second mutation Mating Origin tYPe SM81-11 281-9-2 28 1-9-2 281-9-2 281-9-2 0-42 CP 143 WGG-FA40 Both Both Both matl-1 Both matl-1 mat 1-2 matl-1 First mutation arg-1 lys- I lys-2 ~YS-3 ser-2 asn-2 nic-2 arg-6 ~ arg-1 lys-I lys-2 Iys-3 - + + + - - - ~ ~ ~ ~~~ ser-2 asn-2 nic-2 + + + + + + + - NT NT NT NT NT NT - + NT + - + ~ + - ~ arg-6 + + + - ~~~ Strains carrying the auxotrophic mutations indicated were tested for the ability to form prototrophic heterokaryons. Cases where the strains showed complementation are indicated by a “+” sign. In all cases that showed no complementation (indicated by a =-” sign), at least eight independent trials were conducted. Possible pairings that were not tested for heterokaryosis are indicated by “NT.” All cultures used in these tests originated from either a single ascospore or conidium. The column headed “Origin” indicates the parental strain from which the auxotrophic mutant was originally isolated. T h e column headed “Mating-type tested indicates the mating type of strains that were used in the complementation tests. In those cases where a single mating type is shown (matl-1 or matl-2), the strain tested was the original mutant, as isolated from the parent listed under “Origin.” In the cases where “both” is entered, two strains were tested, one being the original mutant and the other being a strain of the opposite mating type, produced by crossing the original mutant with a strain closely related to the parent of origin, on their failure to complement and their similar growth response to intermediates in lysine biosynthesis (K. PARSONS, unpublished results), we believe they are alleles of a single gene. Thus, we will now designate them as lysl-1, Zysl-2 and lysl-3. T h e mutants carrying arg-1 and arg-6 differ in their growth response to biosynthetic intermediates (K. PARSONS,unpublished results), and they complement, indicating that different genes are affected. We will now designate these mutations as argl-1 and arg2-6. DISCUSSION Different modes of heterokaryotic growth have been described in mycelial fungi. N . crassa forms heterokaryons in which all cells in the mycelium are and COONRADT 1944; PITTENGER and ATWOOD1956; heterokaryotic (BEADLE DAVIS 1966). Conidia and hyphal tip subcultures from heterokaryons of N . crassa are themselves heterokaryotic. Heterokaryons of V. dahliae are quite different. The only binucleate cells are at points of anastomosis 1-2 mm behind and MAYFIELD1974). Intrahyphal the growing edge of the colony (PUHALLA diffusion of small molecules, such as amino acids, from these relatively rare heterokaryotic cells feeds the growing hyphal tip. Conidia and hyphal tip subcultures from such heterokaryons are homokaryotic and exhibit the phenotype of one or the other of the parents. Heterokaryosis and parasexual recombination have been previously reported in M. grzsea (YAMASAKI and NIIZEKI1965; GENOVESI and MACILL 19’76), but these studies did not discriminate between heterokaryon and diploid formation, 1126 M. S . CRAWFORD E T A L . and they did not describe M. grisea heterokaryons in detail. The prototrophic growth that occurs when two M . grisea auxotrophic strains are paired is not the result of cross-feeding, since complementary auxotrophs separated by a dialysis membrane fail to grow. Hyphal tip subcultures from M. grisea heterokaryons are auxotrophs with phenotypes identical to the parental strains. A11 M . grisea conidia from heterokaryotic cultures have the growth requirements of one or the other of the parental strains. All those conidia that we have examined contained only one nucleus per cell; no heterokaryotic conidia were detected. These properties of M . grisea heterokaryons resemble those of V. dahliae heterokaryons; therefore, M . grisea heterokaryotic growth may be fed by binucleate cells at points of anastomosis behind the leading edge of the colony. The similarity between M . grisea and V. dahliae heterokaryons is not surprising since the mycelia produced by these two fungi are similar in having septate cells with one nucleus per cell. Operationally, the properties we have observed for M . grisea heterokaryons define the usefulness of these heterokaryons for genetic analysis of the pathogen. As we have already shown (Table 9), M. grisea heterokaryons will be useful for complementation and dominance testing of mutations that affect nutritional phenotypes. However, straightforward complementation and dominance testing of genes that determine pathogenicity would be impossible using M . grisea heterokaryons, since they produce only conidia of each parental type, and the normal route of infection is through conidia. Fast-growing sectors arising from heterokaryotic colonies are at least transiently diploid, as indicated by the recovery of conidia that show recombination of parental auxotrophic markers. We have isolated rare, unstable prototrophic conidia from diploid cultures that segregate the original markers present in the diploid strain. We have referred to these conidia as diploids because we are able to obtain from them all markers characteristic of the original parental strains; however, these conidia may be aneuploid. The formation and behavior of aneuploids in Aspergallus nidulans was analyzed using diploids marked at numerous loci representing all eight chromosomes (KAFER 1960, 196 1). Such highly sophisticated genetic analysis will be required to further describe diploidy and aneuploidy in vegetative cultures of M . grisea. It appears that the sexual cycle is an unlikely source of variation in nature for M. grisea strains that infect rice. This is because newly acquired isolates of M . grisea that infect rice are uniformly female sterile (ITOI et al. 1983; VALENT et al. 1986), and rice pathogens from a single geographical area appear to be predominantly of the same mating type. Our results support the suggestion that the parasexual cycle is an important source of variation for this fungus in and MAGILL 1976). We have nature (YAMASAKIand NIIZEKI1965; GENOVESI been able to detect both haploidization and mitotic recombination in a very unstable vegetative diploid phase. M . grisea diploids appear to be less stable than those of A. nidulans, the perfect fungus in which extensive characterizaet al. 1953; KAFER tion of the parasexual cycle has been achieved (PONTECORVO 1960, 1961; LHOAS1967). T h e degree of instability we have observed may be M . GRISEA HETEROKARYONS AND DIPLOIDS 1127 more similar to that seen in some imperfect fungi, including the plant pathogen, Verticillium albo-atrum (HASTIE1968). The suggested importance of the parasexual cycle as a source of variation of M . grisea in nature is consistent with our failure to detect interstrain barriers to the formation of heterokaryons or diploids. T h e strains used for this study were derived from Japanese field isolates that infect either finger millet or rice. These field isolates were collected over a period of 20 yr, and at diverse areas within Japan. Our studies have been restricted so far to Japanese strains, but the lack of vegetative incompatibility is nevertheless an interesting observation, since in other fungi, field isolates from the same area often show as much incompatibility as isolates from different areas in the world (MYLYK 1976; ANAGNOSTAKIS and WAGGONER1981). One measure of the relative fertility of M . grisea strains is the frequency of viable ascospores produced in a sexual cross (VALENTet al. 1986). T h e crosses reported here yielded 20-80% viable ascospores depending on the strains involved. We have demonstrated the potential for fertility improvement in M . grisea strains by inbreeding and selection of highly fertile progeny (VALENTet al. 1986). Since the auxotrophic mutants reported here were not isolated from the most fertile strains now available in our laboratory, the potential for genetic analysis of this fungus is even more favorable than these studies might indicate. T h e results reported in this paper suggest that mitotic mapping of genes may be possible in this system. This work was supported by the Department of Energy (DE-AC02-76ERO-1426and DE-ACO284ER13 160), by The Rockefeller Foundation (RF81042) and by Monsanto Agricultural Products Company. We should like to acknowledge the support and encouragement given by PETERALBERSHEIM, in whose laboratory this work was performed. We should also like to thank FRANC= LATTERELL for introducing us to Pyricularia and for supplying fungal cultures; many techniques currently in use in our laboratory were developed by her. We are especially grateful to HAJIME KATO and HIROSHIYAEGASHIfor sharing strains of Pyricularia and for providing helpful insights on the Pyricularia as pathogens. HAJIMEKATO generously arranged field trips for B.V. in Ibaraki and Tochigi Prefectures in Japan during July, 1982, for the purpose of collecting fresh field isolates of the fungus. 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