Mating type and the differentiated state in Physarum polycephalum.

In the acellular slime mold, Physarum polycephalum, the differentiation of amoebae into plasmodia is controlled by a mating type locus, mt. Amoebae carrying heterothallic alleles usually do not differentiate within clones; plasmodia form when two amoebae carrying different alleles fuse and undergo karyogamy. In this paper, we show that amoebae heterozygous for heterothallic alleles can be isolated and maintained as amoebae; the amoebae form plasmodia in clones without a change in ploidy. Plasmodia were also found to be formed, infrequently, by heterothallic amoebae of a single mating type. The plasmodia are healthy and are also formed without a change in ploidy. Thus, the presence of two different heterothallic mating type genes in a single nucleus is compatible with the amoebal state and one heterothallic mating type gene is compatible with the plasmodial state, once established.     

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.     

Building a plasmodium: Development in the acellular slime mould Physarum polycephalum

The two vegetative cell types of the acellular slime mould Physarum polycephalum - amoebae and plasmodia - differ greatly in cellular organisation and behaviour as a result of differences in gene expression. The development of uninucleate amoebae into multinucleate, syncytial plasmodia is under the control of the mating-type locus matA, which is a complex, multi-functional locus. A key period during plasmodium development is the extended cell cycle, which occurs in the developing uninucleate cell. During this long cell cycle, many of the changes in cellular organisation that accompany development into the multinucleate stage are initiated including, for example, alterations in microtubule organisation. Genes have been identified that show cell-type specific expression in either amoebae or plasmodia and many of these genes alter their pattern of expression during the extended cell cycle. With the introduction of a DNA transformation system for P. polycephalum, it is now possible to investigate the functions of genes in the vegetative cell types and their roles in the cellular reorganisations accompanying development.     

Food deprivation is not a prerequisite for the amoebal to plasmodial transition in Physarum polycephalum

The effect of food supply on the onset of asexual and sexual plasmodium formation in Physarum polycephalum was studied. Asexual differentiation occurs readily in amoebae carrying the matAh mating type allele. The density at which these amoebae begin to differentiate is influenced by the ind locus, which controls the production of a diffusible inducer. The alleles ind-1 and ind-2 are known. Strains carring the ind-1 allele begin plasmodium formation at a low amoebal density (rapid differentiation), while strains carring the ind-2 allele differentiate at a higher amoebal density (slow differentiation). The onset of differentiation is characteristic of the strain and did not change with a 20-fold variation in the number of food bacteria available. Sexual differentiation occurs between compatible amoebal strains. For a given pair of amoebal strains the onset of plasmodium formation occurs at a characteristic cell density that is determined by the genetic backgrounds of the strains. The ind locus is one of the genes that influences this cell density. Plasmodia are formed at a lower cell density in crosses involving compatible amoebae carrying the ind-1 allele than they are in crosses with strains carrying the ind-2 allele. As was found for asexual differentiation, an approximate 20-fold variation in the food supply did not affect the initiation of sexual plasmodium formation. These results suggest that in most cases starvation does not trigger the differentiation of amoebae into plasmodia. The time of onset of plasmodium formation is determined largely by genetic factors.     

Mutants with decreased differentiation to plasmodia in Physarum polycephalum

Summary Mutant (APT) amoebae that display reduced ability to form plasmodia asexually were isolated by the use of an enrichment procedure. The results of reconstruction experiments show that the procedure enriches only for mutants blocked early in the pathway from amoeba to plasmodium. Mutants were isolated from four parents, two of which produce plasmodia asexually because they carry the allele mth of the mating type locus, and two because they carry gad (greater asexual differentiation) mutations. The APT mutants varied widely in the frequency of residual plasmodium formation, which occurred, in some cases, by reversion. The mutants, called apt (amoeba to plasmodium transition), were recessive in diploids and linked to the mating type (mt) locus. Mutants derived from the gad parents, unlike the parents themselves, crossed readily with heterothallic amoebae. Progeny analysis from such crosses indicates that both gad mutations are linked to mt. The mutants derived from one of the mth parents fell into two groups on the basis of their ability to cross with the mutants derived from the mt2 gad-8 parent. The result suggests that the mth-derived mutants represent two or more complementation groups. Mutants derived from the mt2 gad-8 parent cross with mt2 amoebae and hence display an altered mating specificity.     

A New Mating Compatibility Locus in PHYSARUM POLYCEPHALUM

The rate and extent of plasmodium formation were studied in mating tests involving pairs of largely isogenic amoebal strains compatible for mating-type (mt) alleles. A systematic variability was observed: plasmodia formed either rapidly and extensively or slowly and inefficiently. Plasmodium formation was found to be 10³- to 10⁴-fold more extensive in "rapid" crosses than in "slow" crosses. A genetic analysis revealed that the variability reflects the influence of a multiallelic compatibility locus that determines mating efficiency. This compatibility locus (designated matB), together with the original mating type locus, mt (in this work designated matA), constitute a tetrapolar mating specificity system in Physarum polycephalum.     

Identification of three genes expressed primarily during development in Physarum polycephalum

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation. Received: 21 June 1999 / Accepted: 17 August 1999     

Revertants of selfing (gad) mutants in Physarum polycephalum

The conversion of the uninucleate amoebal form of Physarum polycephalum to the multi-nucleate plasmodial form is under the control of a genetic region which contains matA (or mt), a determinant of mating specificity. The region is the site of most gad mutations, which give amoebae the ability to produce plasmodia in clones without mating (ie, to self). In the present study, nonselfing revertants were isolated from two matA2-derived gad mutants and two matA3-derived gad mutants. Some revertants were found to have regained exactly, or nearly, the same phenotype as the original matA2 or matA3 strain. Others expressed new mating types, having gained the ability to mate with strains of the parental matA type. The results are compatible with a model in which new mating types arise from forward mutations (gad) and back mutations (npf or no plasmodium formation) occurring successively in a single gene, matA.     

Mutations Increasing Asexual Plasmodium Formation in PHYSARUM POLYCEPHALUM

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 (mt1, mt2, mt3, mt4). At one extreme, only one 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 mutant 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-11 ), 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 amoebal phase of the life cycle. Spores from five of the 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 responsible for the ALC phenotype in this mutant. Like gad-12, alc-1 is expressed with different mt alleles. Preliminary observations 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.     

Two Multiallelic Mating Compatibility Loci Separately Regulate Zygote Formation and Zygote Differentiation in the Myxomycete PHYSARUM POLYCEPHALUM

The mating of Physarum polycephalum amoebae, the ultimate consequence of which is a "plasmodium," was recently shown to be governed by two compatibility loci, matA (or mt) and matB (Dee 1978; Youngmanet al. 1979). We present evidence that matA and matB separately regulate two discrete stages of mating: in the first stage, amoebae (which are normally haploid) fuse in pairs, with a specificity determined by matB genotype, to form diploid zygotes; subsequent differentiation of the zygotes into plasmodia is regulated by matA and is unaffected by matB. Mixtures of amoebae carrying unlike matA and matB alleles formed diploids to the extent of 10 to 15% of the cells present, and the diploids differentiated into plasmodia. When only the matB alleles differed, diploid cells still formed to a comparable (5 to 10%) extent, but rather than differentiating, these diploids remained amoebae. When strains carried the same alleles of matB, formation of diploid cells was greatly reduced: in like-matB, like-matA mixtures, none of 320 cells examined was diploid; in like-matB, unlike mat-A mixtures, differentiating diploids could be detected, but at only 10(-3) to 10(-2) the frequency of unlike-matB, unlike-matA mixtures. The nondifferentiating diploid amoebae recovered from unlike-matB, like-matA mixtures were genetically stable through extensive growth, even though they grew more slowly than haploids (10-hr vs. 8-hr doubling period), and could be crossed with both haploids and diploids. The results of such higher ploidy and mixed ploidy crosses indicate that karyogamy does not invariably accompany zygote formation and differentiation.     

A Gene, ALCA, Affecting the Life Cycle Form Expressed in PHYSARUM POLYCEPHALUM

The usual sequence of forms in the Physarum polycephalum life cycle is plasmodium-spore-amoeba-plasmodium. So-called "amoebaless life cycle" or alc mutants of this Myxomycete undergo a simplified plasmodium-spore-plasmodium life cycle. We have analyzed three independently isolated alc mutants and found in each case that the failure of the spores to give rise to amoebae is due to a recessive Mendelian allele. The three mutations are tightly linked to one another and belong to a single complementation group, alcA. The mutations are pleiotropic, not only interfering with the establishment of the amoebal form at spore germination, but also affecting the phenotype of alc amoebae, which occasionally arise from alc spores. The alc amoebae (1) grow more slowly than wild type, particularly at elevated temperatures; (2) tend to transform directly into plasmodia, circumventing the sexual fusion of amoebae that usually accompanies plasmodium formation; and (3) form plasmodia by the sexual mechanism less efficiently than wild-type amoebae. The various effects of an alc mutation seem to derive from mutation of a single gene, since reversion for one effect is always accompanied by reversion for the other effects. Moreover, a mutation, aptA1, that blocks direct plasmodium formation by alcA amoebae, also increases their growth rate to near normal. The manner of plasmodium formation in alcA strains differs significantly from that in another class of mutants, the gad mutants. Unlike gad amoebae, alcA amoebae need not reach a critical density in order to differentiate directly into plasmodia and do not respond to the extracellular inducer of differentiation. In addition, alcA differentiation is not prevented by a mutation, npfA1, that blocks direct differentiation by most gad amoebae.     

Temperature-sensitive mutants of the slime mould Physarum polycephalum

Summary Methods are described for the isolation and testing of temperature-sensitive plasmodial strains of Physarum polycephalum. Nineteen temperature-sensitive strains were found by screening plasmodia derived from mutagenised amoebae and the properties of these are described. A scheme is outlined for the detection of specific mitotic cycle lesions amongst temperature-sensitive strains, and the properties of a presumptive mitotic cycle mutant are described.     

Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium     

Events in the amoebal-plasmodial transition ofPhysarum polycephalum studied by enrichment for committed cells

Summary Amoebae of strain CLof Physarum polycephalum undergo apogamic development to form multinucleate plasmodia. During the amoebalplasmodial transition, large uninucleate cells become irreversibly committed to plasmodium development. In developing cultures, amoebae lose the ability to flagellate before they become committed. Enriched suspensions of committed cells can be obtained by inducing asynchronous differentiating cultures to flagellate and passing the cells through a glass bead column. Committed cells can be cultured to form plasmodia on bacterial lawns or in axenic liquid medium but cannot be cultured on axenic agar medium. Uninucleate committed cells express tubulin isotypes characteristic of amoebae, but after culture in axenic liquid medium, the cells express plasmodial specific tubulin isotypes.Abbrevations SDM Semi-defined medium - DSDM Dilute semidefined medium - LIA Liver infusion agar - SBS Standard bacterial suspension - IEF Isoelectric focussing - SDS Sodium dodecyl sulphate - PAUF Precommitted amoebae unable to flagellate (for the explanation of these cells see text).     

Apogamic development of plasmodia in the myxomycetePhysarum polycephalum: a cinematographic analysis

Summary Strain CL ofPhysarum polycephalum forms multinucleate plasmodia within clones of uninucleate amoebae. The plasmodia have the same nuclear DNA content as the amoebae. Analysis of plasmodial development, using time-lapse cinematography, showed that binucleate cells were formed as a result of nuclear division in uninucleate cells. Binucleate cells developed into plasmodia by further nuclear divisions and cell fusions. No fusions involving uninucleate cells were observed. A temporary increase in cell and nuclear size occurred at the time of binucleate cell formation.     

The Sequence of Regulatory Events in the Sporulation Control Network of Physarum polycephalum Analysed by Time-resolved Somatic Complementation of Mutants

The developmental decision for sporulation of Physarum polycephalum plasmodia is under sensory control by environmental factors like visible light or heat shock and endogenous signals like glucose starvation. Several hours after perceiving an inductive stimulus, plasmodia become committed to sporulation; thereby, they lose their unlimited replicative potential and execute a developmental program that involves differentiation into various cell types required to form a mature fruiting body. Plasmodia are multinuclear single cells which spontaneously fuse upon physical contact. Fusion of mutant plasmodia and cytoplasmic mixing allows complementation studies to be performed at the functional level. Mutant cells altered in their ability to sporulate in response to phytochrome activation by far-red light were cured by fusion with wild-type or other mutant plasmodia. Phytochrome activation in one plasmodium and subsequent fusion with a non-induced plasmodium revealed that complementation of the two mutations depended on (i) which of two genetically distinct plasmodial cells was stimulated; and (ii) on the delay time elapsed between stimulation and cytoplasmic mixing. Such experiments allow us to determine the kinetics and the causal sequence of the regulatory events tagged by mutation.     

Mutations affecting the initiation of plasmodial development in Physarum polycephalum

In the heterothallic myxomycete Physarum polycephalum, uninucleate amoebae normally differentiate into syncytial plasmodia following heterotypic mating. In order to study the genetic control of this developmental process, mutations affecting the amoebal-plasmodial transition have been sought. Numerous mutants characterized by self-fertility have been isolated. The use of alkylating mutagens increases the mutant frequency over the spontaneous level but does not alter the mutant spectrum. Three spontaneous and 14 induced mutants have been analyzed genetically. In each, the mutation appears to be linked to the mating type locus. In three randomly selected mutants, the nuclear DNA content is the same in amoebae and plasmodia, indicating that amoebal syngamy does not precede plasmodium development in these strains. These results indicate that a highly specific type of mutational event, occurring close to or within the mating type locus, can abolish the requirement for syngamy normally associated with plasmodial differentiation. These mutations help define a genomic region regulating the switch from amoebal to plasmodial growth.     

Complementation of Amoebal-Plasmodial Transition Mutants in PHYSARUM POLYCEPHALUM

Amoebae of the Myxomycete Physarum polycephalum differentiate to yield plasmodia in two ways: in crossing, haploid amoebae of appropriate genotypes fuse to form diploid plasmodia; in selfing, plasmodia form without amoebal fusion or increase in ploidy. Amoebae carrying the mating-type allele matAh (formerly mt_h) self efficiently, but occasionally give rise to mutants that self at very low frequencies. Such "amoebal-plasmodial transition" mutants were mixed in pairs to test their ability to complement one another in the formation of plasmodia by crossing. The pattern of crossing permitted 33 mutants to be assigned to four complementation groups (aptA^-, npfA^-, npfB^- and npfC^-). Similar tests had previously proved only partially successful, as crossing had occurred only rarely in mixtures of compatible strains. The efficiency of complementation was greatly increased in the current work by mixing strains that carried different alleles of a newly-discovered mating-compatibility locus, matB; this locus had no effect on the specificity of complementation. A possible interpretation of the complementation behavior of the mutants is suggested.     

Analysis of development and growth in a mutant of Physarum polycephalum with defective cytokinesis

Cultures of amoebae of the mutant strain ATS23 isolated from strain CLd of Physarum polycephalum contain multinucleate cells and cells with increased nuclear DNA content. Plasmodia derived from ATS23 clones show abnormal morphology and defective sporulation. All abnormalities are enhanced by high incubation temperature (31 °C). Genetic analysis suggested that all the abnormalities were caused by a single mutation, denoted hts-23. The kinetics of plasmodium formation were followed in cultures of apogamic amoebae carrying hts-23 and hts⁺ (wild type) respectively. Results indicated that, relative to wild type, hts-23 did not increase the rate of plasmodium formation. There was evidence that, in both mutant and wild-type strains, commitment to plasmodium development occurred in uninucleate cells. Analysis of cell pedigrees by time-lapse cinematography indicated that the primary abnormal event in cultures of hts-23 amoebae was failure of cytokinesis; an apparently complete cleavage furrow was formed but cell separation failed, resulting in a binucleate cell. This event occurred randomly in pedigrees in which the majority of divisions were completed normally; its frequency increased during incubation at 31 °C. All other abnormalities in hts-23 amoebal cultures could be attributed to this primary event, assuming that DNA synthesis continued in the absence of cytokinesis and that the binucleate cells underwent the amoebal type of “open” mitosis, allowing the possibility of spindle fusion. This implies that the acquisition of “closed” mitosis is an essential early step in plasmodium development.