Morphological and Mutational Analysis of Mating in Ustilago hordei

Martinez-Espinoza, A. D., Gerhardt, S. A., and Sherwood, J. E. 1993. Morphological and mutational analysis of mating in Ustilago hordei. Experimental Mycology 17, 200-214. Ustilago hordei is a basidiomycete that causes covered smut on barley. Mating in U. hordei, which is controlled by a single locus with two alleles, results in the conversion of haploid, nonpathogenic yeast-like sporidia to dikaryotic, pathogenic mycelia. When sporidia of the opposite mating type were mixed and placed on water agar, both cell types produced conjugation tubes within 2 h at 21°C. Growth of conjugation tubes was directed toward the tip of tubes arising from cells of the opposite mating type. These tubes fused and the dikaryotic mycelium emerged from the conjugation bridge. Sporidia separated by a dialysis membrane were still capable of inducing conjugation tube formation by cells of the opposite mating type, indicating the involvement of diffusible small-molecular-weight mating factors (pheromones). Numerous nutritional and environmental variables were examined in order to optimize conjugation tube induction. Twenty-six mutants that fail to form dikaryotic mycelium have been isolated and characterized. These mutants were arranged into classes based on their ability to form conjugation tubes, the ability to induce conjugation tube formation by opposite mating-type cells, and cell morphology. These mutants provide an indication of the genetic complexity involved in this critical phase of the U. hordei life cycle.     

Evidence for Mitochondrial DNA Polymorphism and Uniparental Inheritance in the Cellular Slime Mold Polysphondylium Pallidum: Effect of Intraspecies Mating on Mitochondrial DNA Transmission

Restriction fragment length polymorphisms (RFLPs) were used as markers to monitor mitochondrial inheritance in the cellular slime mold, Polysphondylium pallidum. When two opposite mating types (mat1 and mat2) of closely related strains were crossed, all the haploid progeny regardless of mating type inherited their mitochondrial DNA from the mat2 parent only. When opposite mating types from more distantly related strains were crossed, most of the progeny also inherited their mitochondrial DNA from the mat2 parent, but some inherited their mitochondrial DNA from the mat1 parent. In both cases however, the transmission of mitochondrial DNA was uniparental, since in every individual progeny only one type of mitochondrial DNA exists. Moreover, in crosses involving more distantly related strains all the progeny of a single macrocyst were shown to contain the same type of mitochondrial DNA. These findings are discussed in regard to mechanisms of transmission and the possible involvement of nuclear genes in the control of transmission of mitochondrial DNA in Polysphondylium.     

Asymmetry and directionality in production of new cell types during clonal growth: the switching pattern of homothallic yeast.

Homothallic Saccharomyces yeasts efficiently interconvert between two cell types, the mating types a and alpha. These interconversions have been proposed to occur by genetic rearrangement ("cassette" insertion) at the locus controlling cell type (the mating type locus). The pattern of switching from one cell type to the other during growth of a clone of homothallic cells has been followed by direct microscopic observation, and the results have been summarized as "rules" of switching. First, when a cell divides, it produces either two cells with the same mating type as the original cell or two cells that have switched to the other mating type. This observation suggests that the mating type locus is changed early in the cell cycle, in late Gl or during S. Second, the ability to produce cells that have switched mating type is restricted to cells that have previously divided ("experienced cells"). Spores and buds ("inexperienced cells") rarely if ever give rise to cells with changed mating type. A homothallic yeast cell thus exhibits asymmetric segregation of the potential for mating type interconversion--at each cell division, the mother, but not the daughter, is capable of switching cell types in its next division. Homothallic cells also exhibit directionality in switching: experienced cells switch to the opposite cell type in more than 50% of cell divisions. These results show that the process of mating type interconversion is itself controlled during growth of a clone of homothallic cells. By analogy and extension of these results, we propose that multiple cell types can be produced in a specific pattern during development of a higher eucaryote in a model involving sequential cassette insertion.     

Identification and Characterization of a Mutation Affecting the Division Arrest Signaling of the Pheromone Response Pathway in Saccharomyces Cerevisiae

Mating pheromones, a- and alpha-factors, arrest the division of cells of opposite mating types, alpha and a cells, respectively. I have isolated a sterile mutant of Saccharomyces cerevisiae that is defective in division arrest in response to alpha-factor but not defective in morphological changes and agglutinin induction. The mutation was designated dac2 for division arrest control by mating pheromones. The dac2 mutation was closely linked to gal1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11, ste12, ste18 and dac1). Although dac2 cells had no phenotype in the absence of pheromones, they showed morphological alterations and divided continuously in the presence of pheromones. As a result, dac2 cells had a mating defect. The dac2 mutation could suppress the lethality caused by the disruption of the GPA1 gene (previously shown to encode a protein with similarity to the alpha subunit of mammalian G proteins). In addition, dac2 cells formed prezygotes with wild-type cells of opposite mating types, although they could not undergo cell fusion. These results suggest that the DAC2 product may control the signal for G-protein-mediated cell-cycle arrest and indicate that the synchronization of haploid yeast cell cycles by mating pheromones is essential for cell fusion during conjugation.     

Sexual development in the homothallic green alga Chlamydomonas monoica Strehlow

In Chlamydomonas monoica, cell division and mating are interdependent processes, since under gametogenic conditions only newly born cells are mating competent. By refeeding nitrogen-starved cells with nitrate or ammonium ions, cell division and mating were synchronized. The mating competence of the progeny cells was dependent on the amount of the nitrogen source parent cells were refed, with an optimum around 0.1 mol·10⁵ cells. A second treatment with nitrate inhibited gametogenesis, but only when applied during the first part of the cell cycle, suggesting that an essential part of sexual development takes place during this period. During the latter part of the cell cycle, cells required light to acquire mating competence.     

Ustilago maydis Mating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bolker, M., and Kahmann, R. 1996. Ustilago maydis mating hyphae orient their growth toward pheromone sources. Fungal Genetics and Biology 20, 299-312. When small drops of Ustilago maydis sporidia were placed 100-200 μm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example, a2b2 mating hyphae grew toward a1b1 and a1b2 mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with the a2 allele began elongating before sporidia with the a1 allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous at b but not at a formed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, the a2a2b1b2 strain formed filaments more quickly than the a1a1b1b2 strain. Taken together, these results suggest that the a2 pheromone diffuses less readily or is degraded more quickly than the a1 pheromone.     

Mating-Type-Dependent Inhibition of Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae

Yeast cells of mating type α excrete a sex factor which inhibits cell division and deoxyribonucleic acid replication but not ribonucleic acid or protein synthesis in cells of opposite mating type a.     

Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA

Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation. The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division. On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h. This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium. These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1. Starved cells that had finished division soon formed mating pairs with cells of the opposite type. These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T. thermophila.     

Selfing versus outcrossing propensity of the fungal pathogen Microbotryumviolaceum across Silenelatifolia host plants

In the fungal pathogen Microbotryumviolaceum mating (i.e. conjugation between cells of opposite mating type) is indispensable for infection of its host plant Silenelatifolia. Since outcrossing opportunities are potentially rare, selfing may be appropriate to ensure reproduction. On the other hand, outcrossing may create genetic variability necessary in the coevolutionary arms race with its host. We investigated the propensity of M. violaceum to outcross vs. self in different host environments. We used haploid sporidia from each of three strains from five fungal populations for pairwise mixtures of opposite mating type, representing either selfing or outcrossing combinations. Mixtures were exposed to leaf extract from seven S. latifolia plants. The proportion of conjugated sporidia quantified mating propensity. The identity of both fungal strains and host influenced conjugation. First, individual strains differed in conjugation frequency by up to 30%, and strains differed in their performance across the different hosts. Second, selfing combinations produced, on average, more conjugations than did outcrossing combinations. Selfing appears to be the predominant mode of reproduction in this fungus, and selfing preference may have evolved as a mechanism of reproductive assurance. Third, individual strains varied considerably in conjugation frequency in selfing and outcrossing combinations across different hosts. This indicates that conjugation between outcrossing partners could be favoured at least in some hosts. Since the dikaryon resulting from conjugation is the infectious unit, conjugation frequency may correspond with infection probability. This assumption was supported by an inoculation experiment, where high infectious sporidial dosage resulted in higher infections success than did low dosage. We therefore predict that sexual recombination can provide this pathogen with novel genotypes able to infect local resistant hosts.     

Common sex-linked deleterious alleles in a plant parasitic fungus alter infection success but show no pleiotropic advantage

Microbotryum violaceum is a fungus that causes the sterilizing anther smut disease in Caryophyllaceae. Its diploid teliospores normally produce equal proportions of haploid sporidia of its two mating types. However natural populations contain high frequencies of individuals producing sporidia of only one mating type ('biased strains'). This mating type-ratio bias is caused by deleterious alleles at haploid phase ('haplo-lethals') linked to the mating type locus that can be transmitted only by intra-tetrad selfing. We used experimental inoculations to test some of the hypotheses proposed to explain the maintenance of haplo-lethals. We found a disadvantage of biased strains in infection ability and high intra-tetrad mating rates. Biased strains had no higher competitive ability nor shorter latency and their higher spore production per flower appeared insufficient to compensate their disadvantages. These findings were only consistent with the hypothesis that haplo-lethals are maintained under a metapopulation structure because of high intra-tetrad selfing rates, founder effects and selection at the population level.     

Genetics of Ustilago violacea. XXXV. Transposition in Haploid and Diploid Sporidia and Germinating Teliospores

Ustilago violacea sporidia of the white (w) MAD strain (a-2 w lys-3 ino-1 thi) incubated on minimal medium containing 100 mM KClO3 (potassium chlorate) produced only colonies with the pink phenotype. Sporidia from these colonies retained their pink color on complex medium. Sporidia of the diploid D1 strain (a-1 y nic-1 thi/a-2 w met-1 arg-f Chl70 thi) and of the diploid D2 strain (a-1 y his-1 glu-1 thi/a-2 w lys-3 ino-1 thi) produced pink colonies on complex medium. Streaks of diploid D1 sporidia from the pink colonies were stable on complex medium. In contrast, streaks of diploid D2 sporidia, which are heterozygous for the MAD strain, were unstable, initially producing pink colonies on complex medium but then, with continued incubation, producing white termini. Sporidia from the white termini with diploid morphology continued to yield white colonies. Teliospore colonies from three crosses with the MAD strain as a common parent were uniformly pink or had a pink sector instead of the expected uniformly white colonies or colonies having a white sector. Four of 20 and 13 of 20 teliospore colonies, respectively, from two of the three crosses had both a-1 and a-2 sporidia, and the remaining colonies had only a-1 or only a-2 sporidia. All 40 teliospore colonies from the third cross had only a-1 or only a-2 sporidia. All of these observations indicated that the MAD strain may have two autonomous, transactive transposable elements in different chromosomes and that insertional mutations in at least two haplolethal loci were responsible for the teliospore colonies with only a-1 or only a-2 mating type. Crossing over between a haplolethal locus and the centromere would account for teliospore colonies with both a-1 and a-2 sporidia.     

Gametic differentiation in Chlamydomonas reinhardtii. III. Cell wall lysis and microfilament-associated mating structure activation in wild- type and mutant strains

Cell fusion between mating type plus (mt+) and minus (mt-) gametes of Chlamydomonas reinhardtii is analyzed structurally and subjected to experimental manipulation. Cell wall lysis, a necessary prelude to fusion, is shown to require flagellar agglutination between competent gametes; glutaraldehyde-fixed gametes ("corpses") of one mating type will elicit both agglutination and cell wall lysis in the opposite mating type, whereas nonagglutinating impotent (imp) mutant strains are without effect. The fusion process is mediated by a narrow fertilization tubule which extends from the mt+ gamete and establishes contact with the mt- gamete. Formation of the tubule requires the "activation" of a specialized mating structure associated with the ml+ cell membrane; activation causes microfilaments to polymerize from the mating structure into the growing fertilization tubule. Mating structure activation is shown to depend on gametic flagellar agglutination; isoagglutination mediated by the lectin concanavalin A has no effect. Gametes carrying the imp-l mt+ mutation are able to agglutinate but not fuse with mt- cells; the imp-l gametes are shown to have structurally defective mating structures that do not generate microfilaments in response to gametic agglutination.     

Ustilago maydisMating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bölker, M., and Kahmann, R. 1996.Ustilago maydismating hyphae orient their growth toward pheromone sources.Fungal Genetics and Biology20,299–312. When small drops ofUstilago maydissporidia were placed 100–200 μm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example,a2b2mating hyphae grew towarda1b1anda1b2mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with thea2allele began elongating before sporidia with thea1allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous atbbut not ataformed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, thea2a2b1b2strain formed filaments more quickly than thea1a1b1b2strain. Taken together, these results suggest that thea2pheromone diffuses less readily or is degraded more quickly than thea1pheromone.     

Regulation of mating in the cell cycle of Saccharomyces cerevisiae

The capacity of haploid a yeast cells to mate (fuse with a haploid strain of alpha mating type followed by nuclear fusion to produce a diploid cell) was assessed for a variety of temperature-sensitive cell division cycle (cdc) mutants at the permissive and restrictive temperatures. Asynchronous populations of some mutants do not mate at the restrictive temperature, and these mutants define genes (cdc 1, 4, 24, and 33) that are essential both for the cell cycle and for mating. For most cdc mutants, asynchronous populations mate well at the restrictive temperature while populations synchronized at the cdc block do not. Populations of a mutant carrying the cdc 28 mutation mate well at the restrictive temperature after synchronization at the cdc 28 step. These results suggest that mating can occur from the cdc 28 step, the same step at which mating factors arrest cell cycle progress. The cell cycle interval in which mating can occur may or may not extend to the immediately succeeding and diverging steps (cdc 4 and cdc 24). High frequency mating does not occur in the interval of the cell cycle extending from the step before the initiation of DNA synthesis (cdc 7) through DNA synthesis (cdc 2, 8, and 21), medial nuclear division (cdc 13), and late nuclear division (cdc 14 and 15).     

TIME LAPSE ANALYSES OF SEXUAL REPRODUCTION IN CLOSTERIUM EHRENBERGII (CONJUGATOPHYCEAE)1

The process of sexual differentiation was studied using heterothallic clones of Closterium ehrenbergii Meneghini. The first visible sign of sexual reproduction was agglutination of two or more cells in a group and this was followed by gametangiogenic division and conjugation of gametangial cells. Movements of gametangial cells were carefully studied. Gametangial cells occasionally participated again in gametangiogenesis instead of proceeding directly to the formation of conjugation papilla. The whole process of sexual differentiation from vegetative cell to zygospore was considered to be basically similar in both of the two closely related mating groups, A and B, of C. ehrenbergii. Nevertheless, there were some differences between the two groups in patterns of the sexual differentiation. In Group A, vegetative cell division was completely suppressed by mixing the two complementary mating type clones together into the same medium with high light illumination. This suppression was not caused by the nitrogen depletion in the medium, but by the presence of cells of opposite mating type. In Group B, vegetative cell division and sexual reproduction occurred side by side repeatedly for several days.     

Diverse programs of ascus development in pseudohomothallic species of Neurospora,Gelasinospora, and Podospora

Meiosis and ascospore development in the four-spored pseudohomothallic ascomycetes Neurospora tetrasperma, Gelasinospora tetrasperma, Podospora anserina, and P. fefraspora have been reexamined, highlighting differences that reflect independent origins of the four-spored condition in the different genera. In these species, as in the heterothallic eight-spored N. crassa, fusion of haploid nuclei is followed directly by meiosis and a postmeiotic mitosis. These divisions take place within a single unpartitioned giant cell, the ascus, which attains a length of >0.1 mm before nuclei are enclosed by ascospore walls. Two basically different modes underlie the delivery of opposite mating type nuclei into each of the four ascospores in the different genera. In N. tefrasperma on the one hand, the mating type locus is closely centromere-linked. Mating types therefore segregate at the first meiotic division. The second division spindles of N. tefrasperma overlap and are usually parallel to one another, in contrast to the their tandem arrangement in N. crassa. As a result, nonsister nuclei of opposite mating type are placed close together in each half-ascus and a pair is enclosed in each ascospore. In the Podospora and Gelasinospora species on the other hand, the second-division spindles are in tandem, with sister nuclei of opposite mating type associated as a pair in each half-ascus. It is established for P. anserina and inferred for P. fetraspora and G. fefrasperma that a single reciprocal crossing over almost always occurs in the mating type-centromere interval, ensuring that mating types segregate at the second meiotic division and that nuclei of opposite mating type are enclosed in each ascospore. Other differences are also seen that are less fundamental. Neurospora tetrasperma differs from the other species in the orientation of chromosomes and spindle pole body plaques at interphase (I.) Third-division spindles are oriented parallel to the ascus wall in Gelasinospora but across the ascus in Podospora and Neurospora. The two Podospora species differ from one another in nuclear behavior following mitosis in the young ascospores. In P. tefraspora, two of the four nuclei migrate into the tail cell, which degenerates, leaving one functional nucleus of each mating type. In P. anserina, by contrast, only one of the four nuclei moves into the tail cell, leaving the germinating ascospore with two functional nuclei of one mating type and one of the other. The pseudohomothallic condition with its heterokaryotic vegetative phase has significant consequences for both the individual organism and the breeding system. Genetic controls of development and recombination are complex. Inbreeding is not obligatory. © 1994 WiIey-Liss, Inc.     

Conjugation in Tetrahymena: the relationship between the division cycle and cell pairing

Conjugation, a sexual stage in the life cycle of Tetrahymena, is marked by the pairing of two cells of opposite mating types. Pairing establishes cytoplasmic continuity between the two cells and initiates the complex of nuclear events involved in sexual exchange. After mixing cells of opposite mating types in nonnutrient medium, a 3-hr refractory period ensues before pairing begins.A wave of cell division occurs concurrently with the onset of pairing. However, although all cells pair, the population does not double. This indicates that some cells do not divide and yet are capable of pairing. Apparently division per se is not required for pairing but does occur in most of the cells.Autoradiographic analysis demonstrates that the cells that divide before pairing were at a stage in the cell cycle beyond the initiation of macronuclear replication at the time they were transferred to nonnutrient medium. Cells that did not divide were in G₁ at the time of shift-down. Thus, neither replication nor division is required to be able to fuse. However, since fusion occurs only in G₁ and most cells are not in G₁ at the time of shift-down, a traverse of the cell cycle is required.Shift-down induces G₁ arrest and preparations for the mating reaction. Mixing the cells induces a synchronous wave of division for cells beyond the $\text{G}{}_1\text{S}$ interface. Preparations for the mating reaction occur independently of but simultaneous with the preparations for cell division.     

Microtubule motor protein Kar3 is required for normal mitotic division and morphogenesis in Candida albicans

The kinesin-related protein Kar3 is a minus end-directed molecular motor that plays a multifunctional role in microtubule-directed nuclear movement. Previously, it was shown that Candida albicans Kar3p is critical for nuclear fusion during mating as kar3 mutants were defective in karyogamy. In this study, we confirm that Kar3p is required for nuclear congression in mating but that neither Kar3p nor the dynein motor protein Dyn1p is required for nuclear migration in the mating projection prior to cell fusion. In addition, we show that C. albicans Kar3p plays an important role in the cell and colony morphology of mitotically dividing cells, as evidenced by diminished filamentation of kar3 cells on Spider medium and an increased tendency of mutant cells to form pseudohyphal cells in liquid culture. Loss of Kar3p also led to defects in nuclear division, causing cells to grow slowly and exhibit reduced viability compared to wild-type cells. Slow growth was due, at least in part, to delayed cell cycle progression, as cells lacking Kar3p accumulated in anaphase of the cell cycle. Consistent with a role in mitotic division, Kar3 protein was shown to localize to the spindle pole bodies. Finally, kar3 cells exhibited unstable or aberrant mitotic spindles, a finding that accounts for the delay in cell cycle progression and decreased viability of these cells. We suggest that the altered morphology of kar3 cells is a direct consequence of the delay in anaphase, and this leads to increased polarized growth and pseudohypha formation.     

Relation between the efficiency of homothallic switching of yeast mating type genes and the distribution of cell types.

Homothallic switching of yeast mating type genes occurs as often as each cell division, so that a colony derived from a single haploid spore soon contains an equal number of MATa and MAT alpha cells. Cells of opposite mating types conjugate, and eventually the colony contains only nonmating MATa/MAT alpha diploids. Mutations that reduce the efficiency of homothallic MAT conversions yield colonies that still contain many haploid cells of the original spore mating type plus a few recently generated cells of the opposite mating type. These (a greater than alpha)- or (alpha greater than a)-mating colonies also contain some nonmating diploid cells. As an alternative to microscopic pedigree analysis to determine the frequency of mating type conversions in a variety of mutant homothallic strains, we analyzed the proportions of MATa, MAT alpha, and MATa/MAT alpha cells in a colony by examining the mating phenotypes of subclones. We developed a mathematical model that described the proportion of cell types in a slow-switching colony. This model predicted that the proportion of nonmating cells would continually increase with the size (age) of a colony derived from a single cell. This prediction was confirmed by determining the proportion of cell types in colonies of an HO swi1 strain that was grown for different numbers of cell divisions. Data from subcloning (a greater than alpha) and (alpha greater than a) colonies from a variety of slow-switching mutations and chromosomal rearrangements were used to calculate the frequency of MAT conversions in these strains.     

Fidelity of conjugation in Saccharomyces cerevisiae.

Summary An efficient method for the production of synchronous zygotes in Saccharomyces cerevisiae is described. Cells were synchronised under defined conditions in either an a, mixed culture or by incubation of each mating type in cell-free medium in which cells of the opposite mating type had been grown. Synchronised cells were allowed to fuse under defined conditions on filter membranes. This method was used to test the fidelity of conjugation in S. cerevisiae. Under conditions where cells of a or mating type were in contact with up to 6 cells of each of two strains of opposite mating type, less than 1 multiple mating in 10⁴ diploid matings occurred. It is concluded that in sexual conjugation in S. cerevisiae some process distinct from cell contact restricts cell fusion to paired combinations of conjugant cells.