MUTATIONS INCREASING ASEXUAL PLASMODIUM FORMATION IN PHYSARUM POLYCEPHALUM PAUL N. ADLERI AND CHARLES E. HOLT Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Manuscript received May 9, 1977 Revised copy received July 28, 1977 ABSTRACT Rare plasmodia formed in clones of heterothallic amoebae were analyzed in a search for mutations affecting plasmodium formation. The results show that the proportion of mutants varies with both temperature (18", 26" or 30") and mating-type allele (mti, mt2, mt3, mt4). At one extreme, only me of 33 plasmoida formed by mt2 amoebae at 18" is mutant. At the other extreme, three of three plasmodia formed by mt1 amoebae at 30" are mutant. The mu- tant plasmodia fall into two groups, the GAD (greater asexual differentiation) mutants and the ALC (amoebaless life cycle) mutants. The spores of GAD mutants give rise to amoebae that differentiate into plasmodia asexually at much higher frequencies than normal heterothallic amoebae. Seven of eight gad mutations analyzed genetically are linked to mt and one (gad-12) is not. The gad-12 mutation is expressed in strains with different alleles of mt. The frequency of asexual plasmodium formation is heat sensitive in some (e.g., mt3 gad-li), heat-insensitive in two (mt2 gad-8 and mt2 gad-9) and cold-sensitive in one (mt1 gad-12) of twelve GAD mutants analyzed phenotypically. The spores of ALC mutants give rise to plasmodia directly, thereby circumventing the amoeba1 phase of the life cycle. Spores from five 3f die seven ALC mutants give rise to occasional amoebae, as well as plasmodia. The amoebae from one of the mutants carry a mutation (alc-1) that is unlinked to mt and is respon- sible for the ALC phenotype in this mutant. Like gad-12, ah1is expressed with different mt alleles. Preliminary observatims with amoebae from the other four ALC mutants suggest that two are similar to the one containing alc-1; one gives rise to revertant amoebae, and one gives rise to amoebae carrying an alc mutation and a suppressor of the mutation. PHYSARUM POLYCEPHALUM is an acellular slime mold or Myxomycete. P,n the life cycle of this organism, plasmodia give rise to spores, spores germi- nate to yield amoebae, and amoebae form plasmodia (GRAYand ALEXOPOULOS 1968). Plasmodia are pigmented, vegetative structures in which nuclear di- vision but not cytokinesis occurs; thus, they are typically multinucleate and are of indefinite size. When starved in the light, a plasmodium gives rise to groups of spores borne on stalks. Spores, when moistened, yield amoebae. Amoe- bae are nonpigmented and uninucleate, undergo cytokinesis, and are usually Present address: Department of Biology, University of Virginia, Charlottesville, Virginia 22901. Genetics 87: 401-410 November, 1977. 402 P. N. ADLER AND C. E. HOLT haploid. Like plasmodia, they are vegetative cells that may be grown without change in form for an indefinite period. A multiallelic mating-type locus (mt) controls the mode and frequency of plasmodium formation. Twelve heterothallic alleles (mtl,mt2, . mtl2) and one “selfing” allele (mth) are known (DEE 1966; WHEALS1970; COLLINS 1975; COLLINSand TANG1977). The alleles mth, mtl, mt2, mt3 and mt4 are available in a common genetic background (COOKEand DEE 1975; ADLERand HOLT1974b). In the sexual mode of plasmodium formation, cellular and nu- clear fusion of haploid amoebae carrying diff erent mt alleles produces a diploid plasmodium. Meiosis accompanies sporulation in such a plasmodium (ALDRICH 1967; LAANEand HAUGLI1976). In the asexual mode, plasmodia form within clones of amoebae without evidence of amoebal fusion (ANDERSON,COOKE and DEE 1976). Almost all the nuclei of a plasmodium derived asexually from a haploid amoeba are haploid (COOKEand DEE 1974; ADLERand HOLT1975); however, there are occasional diploid nuclei in such a plasmodium, and it is likely that only these survive sporulation (LAFFLERand DOVE1977). Sexual plasmodium formation is efficient between strains carrying any pair of mating- type alleles, with the exception that mating between mth and mt2 strains is very inefficient (ADLERand HOLT1974b; COOKEand DEE 1975; DAVIDOWand HOLT1977). Asexual plasmodium formation, or selfing, occurs at a high fre- quency in mth amoebae and at much lower frequencies in heterothallic amoebae. In a culture of mth amoebae, more than 10% of the cells become individual plasmodia (YOUNGMANet al. 1977). As shown earlier (ADLERand HOLT1975) and confirmed below, the most frequent selfing in heterothallic strains generates only one plasmodium in lo8 amoebae. Diploid amoebae heterozygous for mt have been isolated; these form diploid plasmodia at a high frequency (ADLER and HOLT1975). Note that although mt has a marked influence on the con- versioln from the amoebal to the plasmodial state, any genotype at mt seems to be compatible with either state. Mutational analysis has revealed additional genes that control whether or not an amoeba becomes committed to the plasmodial state. Mutations at aptA (WHEALS1973) and npfA block the asexual conversion of mth amoebae. The two loci are unlinked to mt and to one another (ANDERSONand DEE 1977). A num- ber of other mutations that interfere with asexual plasmodium are tightly linked to mt; the mutations fall into two complementation groups (ANDERSONand DEE 1977; DAVIDOWand HOLT1977). In the present work, we sought mutations that would increase the frequency of asexual plasmodium formation in heterothallic amoebae. Our method was to pick, grow and sporulate the rare plasmodia formed by heterothallic amoebae and to examine amoebal progeny of the plas- modia for mutant characteristics. The formation of nonmutant plasmodia by the amoebae created a background that seriously interfered with the search. We found that the background could be reduced by appropriate selection of mating type and temperature, and we isolated and characterized 20 mutants. DIFFERENTIATION MUTANTS IN PHYSARUM 403 TABLE 1 Strains Strain number Relevant genotype Reference CHI mth fusA2 fusel COOKEand DEE1975 CH9 mth fusA2 fusC1 aptAI WHEALS1970 CH21 mt3 fusA2 fusC2 ADLERand HOLT1974b CH54 mt3 fusA2 fusC2 ADLERand HOLT1974b CH190 mt3 fusA2 fuse2 ADLERand HOLT1974b CH269 mtl fusA2 fusC1 COOKEand DEE 1975 CH273 mt4 fusA2 fusC2 ADLERand HOLT1974b CH274 mt2 fusAl fuel COOKEand DEE1975 CH322 mt3 actE9 emeE4 fusA2 fuse2 ADLER1975 CH326 mt4 actE8 fusA2 fusC2 ADLER1975 CH3.29 mt4 actE3 fusA2 fusC2 ADLER1975 CH343 mt3 ADLER1975 CH344. mt3 ADLER1975 CH347 mt3 fusA2 fusC2 ADLERand HOLT197413 CH348 mt3 jusA2 fusC2 ADLERand HOLT1974b CH351 mt3 fusA2 fusC2 ADLERand HOLT1974b CH393 mt2 ADLER1975 CH394 mt2 fusAl fusC1 ADLER1975 CH396 mt2 fusA2 fusC1 ADLER1975 CH50l mt4 fusA2 fmC2 ADLER1975 CH586 mt3/mt4 ADLERand HOLT1975 CH6348 mt2 ADLER1975 MATERIALS AND METHODS Media: Plasmodial rich medium agar (PRM-agar), dilutc plasmodial rich medium agar (dPRM-agar), dilute plasmodial rich medium agar pH 7 (dPRM pH 7 agar), and liver infusion agar (LIA) were prepared as described previously (ADLERand HOLT1974b; YOUNGMANer al. 1977). Strains: Strain numbers, with relevant genotype and history-, are shown in Table 1. The fus genes, which affect plasmodial fusion, were used as markers; they do not influence plasmodium formation. All strains have a Colonia (CHI) genetic background. Strain CH21 would be expected to contain about 87.5% Coloiiia genes. All other strains should contain more than 95% Colonia genes. Strain CHI has been called CL previously (ADL.ERand HOLT1974b). Strains CH269 and CH274 are LU648 and LU688, respectively, of COOKEand DEE (1 975). The GAD mutants have the following parents: mutant CH403 (parent CH396) ; CH404 (CH274) ; CH405 (CH394) ; CH4.78 (CH.269) ; CH479 (CH322) ; CH480 (CH394) ; CH484. (CH329) ; CH485 (CH322) ; CH486 (CH394); CH487 (CH394) ; CH489 (CH322); CH496 (CH21), and CH526 (CH326). Mutant CH485 (mt3 gad-7) was isolated at 26"; see Table 5 f3r the other GAD mutants. The ALC mutants have the following parents and temperatures of isolation: mutant CH5001 (parent CH21, 26"); CH5002 (CH269, 30"); CH5003 (CH269, 30"); CH5004 (CH322, 30"); CH5005 (CH326,30°) ; CH5006 (CH326, 30°), and CH5007 (CH326,26"). Culture procedures: Plasmodia were formed from amoebae on dPRM-agar, grown on PRM- agar, and induced to sporulate as described prcviously (ADLERand HOLT1974). Methods for germinating spores and growing amoebae are described in the same publication. Kinetic experiments (YOUNGMANet al. 1977) were performed as follows. Replicate amoeba1 cultures were set up by pipetting 0.05 ml of a suspension of plasmodia-free amoebae and E. coli 404 P. N. ADLER AND C. E. HOLT onto a dPRM agar plate. Cultures were assayed by flooding a plate with 5 ml H,O and scraping the surface of the agar with a glass rod. The resulting suspeiision was diluted with H,O and dilutions plated onto assay plates. Assay plates were either LIA or dPRM pH 7 agar (both of which retarded the differentiation of amoebae into plasmodia) and were incubated at 26" or 30" (this depended on the specific strain) for 5-9 days, and the number of amoebal and plasmodial plaques were counted. For the lowest dilution, 1/5 of the original suspension was plated; thus, the assay should detect as few as 5 plasmodia per original plate. Isolation of plasmodia from heterothallic amoebae: A suspensioii of bacteria and approxi- mately IO4 ameobae was pipetted onto each of a series of dRPM-agar plates. 'The inoculum was allowed to remain as a spot with a diameter of about 20 mm. The plates were incubated in plastic